Cells Useful for Immuno-Based Botulinum Toxin Serotype A Activity Assays

ABSTRACT

The present specification discloses clonal cell lines susceptible to BoNT/A intoxication, methods of producing such clonal cell lines, and methods of detecting Botulinum toxin serotype A activity using such clonal cell lines.

This application is a divisional and claims priority pursuant to 35U.S.C. §120 to U.S. patent application Ser. No. 12/722,801, filed Mar.12, 2010, which claims priority pursuant to 35 U.S.C. §119(e) to U.S.Provisional Patent Application Ser. No. 61/160,199 filed Mar. 13, 2009,both incorporated entirely by reference.

The sequences disclosed in the present specification are contained inthe Sequence Listing submitted with the present specification which ishereby incorporated by reference in its entirety.

The ability of Clostridial toxins, such as, e.g., Botulinum neurotoxins(BoNTs), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G, andTetanus neurotoxin (TeNT), to inhibit neuronal transmission are beingexploited in a wide variety of therapeutic and cosmetic applications,see e.g., William J. Lipham, Cosmetic and Clinical Applications ofBotulinum Toxin (Slack, Inc., 2004). Clostridial toxins commerciallyavailable as pharmaceutical compositions include, BoNT/A preparations,such as, e.g., BOTOX® (Allergan, Inc., Irvine, Calif.),DYSPORT®/RELOXIN®, (Ipsen Ltd., Slough, England), PURTOX® (Mentor Corp.,Santa Barbara, Calif.), XEOMIN® (Merz Pharmaceuticals, GmbH., Frankfurt,Germany), NEURONOX® (Medy-Tox, Inc., Ochang-myeon, South Korea), BTX-A(Biogen-tech Ltd., University, Yantai, Shandong, China); and BoNT/Bpreparations, such as, e.g., MYOBLOC®/NEUROBLOC® (SolsticeNeurosciences, Inc., South San Francisco, Calif.). As an example, BOTOX®is currently approved in one or more countries for the followingindications: achalasia, adult spasticity, anal fissure, back pain,blepharospasm, bruxism, cervical dystonia, essential tremor, glabellarlines or hyperkinetic facial lines, headache, hemifacial spasm,hyperactivity of bladder, hyperhidrosis, juvenile cerebral palsy,multiple sclerosis, myoclonic disorders, nasal labial lines, spasmodicdysphonia, strabismus and VII nerve disorder.

At present the mouse LD₅₀ bioassay, a lethality test, remains the “goldstandard” test used by all pharmaceutical manufacturers to express thepotency of their preparations. S. S. Arnon et al., JAMA 285: 1059-1070(2001). In fact, the units on the pharmaceutical preparations' labelsare mouse LD₅₀ units and the number of animals needed to producestatistically useful LD₅₀ data is large. The advantage of the mouse LD₅₀bioassay is that it measures all the steps necessary for botulinum toxinuptake (e.g., toxin binding to a cell surface receptor, internalizationof the toxin-receptor complex, light chain translocation into thecytoplasm, light chain cleavage of substrate), instead of merelydetermining the activity for only part of this intoxication process,such as, e.g., in vitro assays that only measure light chain enzymaticactivity. Unfortunately, the mouse LD₅₀ bioassay suffers from manydrawbacks including high operational cost due to the large numbers oflaboratory animals required, a lack of specificity since all BoNTserotypes will cause the same measurable end-point, and the potentialfor inaccuracy unless large animal groups are used. In addition, animalrights groups have exerted pressure on regulatory agencies in the UnitedStates (FDA/NICEATM/ICCVAM) and Europe (MHRA and EDQM), and onpharmaceutical companies manufacturing botulinum neurotoxin products toreduce animal testing and more importantly replace the mouse LD₅₀bioassay for product release. The regulatory agencies are engagingpharmaceutical companies to apply the three “Rs” principle to thepotency testing of botulinum neurotoxins: Reduce, Refine, Replace. D.Straughan, Progress in Applying the Three Rs to the Potency Testing ofBotulinum Toxin Type A, Altern. Lab. Anim. 34(3): 305-313 (2006). Inrecent years, several steps have been already taken to reduce and refinethe mouse LD₅₀ bioassay in order to standardize the protocol and producemore consistent data using fewer animals per assay.

Thus, a simple, reliable, validated and governmental agency acceptablebotulinum toxin activity assay that can evaluate the integrity of allthe steps necessary in botulinum toxin uptake would be of significantvalue because such a non-animal based assay would alleviate the need foranimal testing and all the disadvantages, costs and ethical concernsassociated with this type of animal-based assay. Companion patentapplication Ester Fernandez-Salas, et al., Immuno-Based Botulinum ToxinSerotype A Activity Assays, U.S. patent application Ser. No. 12/403,531,provides novel compositions, cells, and methods for assaying theactivity of a BoNT/A useful for various industries, such as, e.g., thepharmaceutical and food industries. Such compositions, cells, andmethods do not use live animals or tissues taken from live animals, butcan evaluate all the steps necessary for neurotoxin action, namely,binding and cellular uptake of toxin, translocation into the cellcytosol, and protease activity.

One of the necessary components for the methods disclosed in U.S. patentapplication Ser. No. 12/403,531 is the use of cells from an establishedcell line that are susceptible to low levels of BoNT/A intoxicationbecause the amount of BoNT/A contained within a commercially-availablepharmaceutical composition is very low. For example, approximately 4-5ng of a BoNT/A complex is contained in the pharmaceutical compositionsold as BOTOX®. Thus, there is a need to identify and create establishedcell lines comprising cells that are susceptible to BoNT/A intoxicationwhen only very low levels of the neurotoxin is present in the sample.The present specification provides novel cells and cell compositions.that are susceptible to BoNT/A intoxication when only very low amountsof BoNT/A is present and thus allow assaying of commercially-availablepharmaceutical composition comprising BoNT/A.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the current paradigm of neurotransmitterrelease and Clostridial toxin intoxication in a central and peripheralneuron. FIG. 1A shows a schematic for the neurotransmitter releasemechanism of a central and peripheral neuron. The release process can bedescribed as comprising two steps: 1) vesicle docking, where thevesicle-bound SNARE protein of a vesicle containing neurotransmittermolecules associates with the membrane-bound SNARE proteins located atthe plasma membrane; and 2) neurotransmitter release, where the vesiclefuses with the plasma membrane and the neurotransmitter molecules areexocytosed. FIG. 1B shows a schematic of the intoxication mechanism fortetanus and botulinum toxin activity in a central and peripheral neuron.This intoxication process can be described as comprising four steps: 1)receptor binding, where Clostridial toxin binding to a Clostridialreceptor complex and initiates the intoxication process; 2) complexinternalization, where after toxin binding, a vesicle containing atoxin/receptor system complex is endocytosed into the cell; 3) lightchain translocation, where multiple events are thought to occur,including changes in the internal pH of the vesicle, formation of achannel pore comprising the H_(N) domain of Clostridial toxin heavychain, separation of the Clostridial toxin light chain from the heavychain, and release of the light chain and 4) enzymatic targetmodification, where the light chain of Clostridial toxin proteolyticallycleaves its target SNARE substrates, such as, e.g., SNAP-25, VAMP orSyntaxin, thereby preventing vesicle docking and neurotransmitterrelease.

FIG. 2 shows volcano plots of genes that are more than 4-fold(log₂(4)=2) up or down regulated in BB10 and H1 cell lines as comparedto the 2D6 cell line. FIG. 2A shows the genes that are up regulated inthe BB10 cell line as compared to the 2D6 cell line. FIG. 2B shows thegenes that are up regulated in the H1 cell line as compared to the 2D6cell line. FIG. 2C shows the genes that are up regulated in the BB10cell line as compared to the parental SiMa cell line (PA). FIG. 2D showsthe genes that are up regulated in the H1 cell line as compared to theparental SiMa cell line (PA). FIG. 2E shows the genes that are upregulated in the parental SiMa cell line (PA) as compared to the 2D6cell line. FIG. 2F shows the genes that are down regulated in the BB10cell line as compared to the 2D6 cell line. FIG. 2G shows the genes thatare down regulated in the H1 cell line as compared to the 2D6 cell line.FIG. 2H shows the genes that are down regulated in the parental SiMacell line (PA) as compared to the 2D6 cell line. Each dot represents aprobe set. The dashed line marks the 95% confidence interval, p≦0.05.Most of the genes that are over expressed in BB10 and H1 compared to 2D6(A and B) are also over or under-expressed in BB10 compared to PA (C),in H1 compared to PA (D), and in PA compared to 2D6 (E), however anumber of them are less than 4-fold over-expressed and/or do not passthe 95% confidence interval.

FIG. 3 shows normalized BIAcore SPR curves of 7.8 nM of the antibodies2E2A6, 1D3B8, 3C1A5 and 2C9B10 and commercial MC-6050 and MC-6053. FIG.3A shows the normalized data for the on-rate of each antibody. FIG. 3Bshows the normalized data for the off-rate of each antibody.

DETAILED DESCRIPTION

The present specification provides novel cells and cell compositionsthat are susceptible to BoNT/A intoxication to allow assaying of BoNT/Aand BoNT/A compositions such as, e.g., commercially-availablepharmaceutical composition comprising BoNT/A. The cell and cellcompositions disclosed in the present specification are useful toconduct methods that can detect picomolar quantities of BoNT/A in asample, such as the methods disclosed in, e.g., Ester Fernandez-Salas,et al., Immuno-Based Botulinum Toxin Serotype A Activity Assays, U.S.patent application Ser. No. 12/403,531, which is hereby incorporated byreference in its entirety. The cells and cell compositions, and theiruse in methods of detecting BoNT/A activity reduce the need for animaltoxicity studies, yet serve to analyze the multiple steps of BoNT/Aintoxication, namely, binding and cellular uptake of toxin,translocation into the cell cytosol, and protease activity. As discussedfurther below, the novel cells and cell compositions of the presentspecification can be used in assays that analyze crude and bulk samplesas well as highly purified di-chain toxins and formulated toxin productsand further are amenable to automated high throughput assay formats.

Thus, aspects of the present specification provide a cell compositioncomprising cells from a clonal cell line that are susceptible to BoNT/Aintoxication.

Other aspects of the present specification provide a method of detectingBoNT/A activity using the clonal cell lines disclosed in the presentspecification.

Clostridia toxins produced by Clostridium botulinum, Clostridium tetani,Clostridium baratii and Clostridium butyricum are the most widely usedin therapeutic and cosmetic treatments of humans and other mammals.Strains of C. botulinum produce seven antigenically-distinct serotypesof botulinum toxins (BoNTs), which have been identified by investigatingbotulism outbreaks in man (BoNT/A, BoNT/B, BoNT/E and BoNT/F), animals(BoNT/C1 and BoNT/D), or isolated from soil (BoNT/G). While all sevenbotulinum toxin serotypes have similar structure and biologicalproperties, each also displays heterogeneous characteristics, such as,e.g., different pharmacological properties. In contrast, tetanus toxin(TeNT) is produced by a uniform group of C. tetani. Two other species ofClostridia, C. baratii and C. butyricum, also produce toxins similar toBoNT/F and BoNT/E, respectively.

Clostridial toxins are each translated as a single chain polypeptide ofapproximately 150 kDa that is subsequently cleaved by proteolyticscission within a disulfide loop by a naturally-occurring protease, suchas, e.g., an endogenous Clostridial toxin protease or anaturally-occurring protease produced in the environment. Thisposttranslational processing yields a di-chain molecule comprising anapproximately 50 kDa light chain (LC) and an approximately 100 kDa heavychain (HC) held together by a single disulfide bond and noncovalentinteractions. Each mature di-chain molecule comprises three functionallydistinct domains: 1) an enzymatic domain located in the LC that includesa metalloprotease region containing a zinc-dependent endopeptidaseactivity which specifically targets core components of theneurotransmitter release apparatus; 2) a translocation domain containedwithin the amino-terminal half of the HC(H_(N)) that facilitates releaseof the LC from intracellular vesicles into the cytoplasm of the targetcell; and 3) a binding domain found within the carboxyl-terminal half ofthe HC(HO that determines the binding activity and binding specificityof the toxin to the receptor complex located at the surface of thetarget cell.

The binding, translocation and enzymatic activity of these threefunctional domains are all necessary for toxicity. While all details ofthis process are not yet precisely known, the overall cellularintoxication mechanism whereby Clostridial toxins enter a neuron andinhibit neurotransmitter release is similar, regardless of serotype orsubtype. Although the applicants have no wish to be limited by thefollowing description, the intoxication mechanism can be described ascomprising at least four steps: 1) receptor binding, 2) complexinternalization, 3) light chain translocation, and 4) enzymatic targetmodification (FIG. 1). The process is initiated when the H_(C) bindingdomain of a Clostridial toxin binds to a toxin-specific receptor systemlocated on the plasma membrane surface of a target cell. The bindingspecificity of a receptor complex is thought to be achieved, in part, byspecific combinations of gangliosides and protein receptors that appearto distinctly comprise each Clostridial toxin receptor complex. Oncebound, the toxin/receptor complexes are internalized by endocytosis andthe internalized vesicles are sorted to specific intracellular routes.The translocation step appears to be triggered by the acidification ofthe vesicle compartment. This process seems to initiate importantpH-dependent structural rearrangements that increase hydrophobicity,promote pore formation, and facilitate separation of the heavy and lightchains of the toxin. Once separated, the light chain endopeptidase ofthe toxin is released from the intracellular vesicle into the cytosolwhere it appears to specifically target core components of theneurotransmitter release apparatus. These core proteins,vesicle-associated membrane protein (VAMP)/synaptobrevin,synaptosomal-associated protein of 25 kDa (SNAP-25) and Syntaxin, arenecessary for synaptic vesicle docking and fusion at the nerve terminaland constitute members of the soluble N-ethylmaleimide-sensitivefactor-attachment protein-receptor (SNARE) family. BoNT/A and BoNT/Ecleave SNAP-25 in the carboxyl terminal region, releasing a nine ortwenty six amino acid fragment, respectively, and BoNT/C1 also cleavesSNAP-25 near the carboxyl terminus releasing an eight amino acidfragment. The botulinum serotypes BoNT/B, BoNT/D, BoNT/F and BoNT/G, andtetanus toxin, act on the conserved central portion of VAMP, and releasethe amino terminal portion of VAMP into the cytosol. BoNT/C1 cleavessyntaxin at a single site near the cytosolic membrane surface. Theselective proteolysis of synaptic SNAREs accounts for the block ofneurotransmitter release caused by Clostridial toxins in vivo. The SNAREprotein targets of Clostridial toxins are common to exocytosis in avariety of non-neuronal types; in these cells, as in neurons, lightchain peptidase activity inhibits exocytosis, see, e.g., Yann Humeau etal., How Botulinum and Tetanus Neurotoxins Block NeurotransmitterRelease, 82(5) Biochimie. 427-446 (2000); Kathryn Turton et al.,Botulinum and Tetanus Neurotoxins: Structure, Function and TherapeuticUtility, 27(11) Trends Biochem. Sci. 552-558. (2002); Giovanna Lalli etal., The Journey of Tetanus and Botulinum Neurotoxins in Neurons, 11(9)Trends Microbiol. 431-437, (2003).

Aspects of the present disclosure comprise, in part, a cell from anestablished cell line. As used herein, the term “cell” refers to anyeukaryotic cell susceptible to BoNT/A intoxication by a BoNT/A or anyeukaryotic cell that can uptake a BoNT/A. The term cell encompassescells from a variety of organisms, such as, e.g., murine, rat, porcine,bovine, equine, primate and human cells; from a variety of cell typessuch as, e.g., neuronal and non-neuronal; and can be isolated from orpart of a heterogeneous cell population, tissue or organism. As usedherein, the term “established cell line” is synonymous with “immortalcell line,” or “transformed cell line” and refers to a cell culture ofcells selected for indefinite propagation from a cell population derivedfrom an organism, tissue, or organ source. By definition, an establishedcell line excludes a cell culture of primary cells. As used herein, theterm “primary cells” are cells harvested directly from fresh tissues ororgans and do not have the potential to propagate indefinitely. Anestablished cell line can comprise a heterogeneous population of cellsor a uniform population of cells. An established cell line derived froma single cell is referred to as a clonal cell line. An established cellline can be one whose cells endogenously express all component necessaryfor the cells to undergo the overall cellular mechanism whereby a BoNT/Aproteolytically cleaves a SNAP-25 substrate and encompasses the bindingof a BoNT/A to a BoNT/A receptor, the internalization of theneurotoxin/receptor complex, the translocation of the BoNT/A light chainfrom an intracellular vesicle into the cytoplasm and the proteolyticcleavage of a SNAP-25. Alternatively, an established cell line can beone whose cells have had introduced from an exogenous source at leastone component necessary for the cells to undergo the overall cellularmechanism whereby a BoNT/A proteolytically cleaves a SNAP-25 substrateand encompasses the binding of a BoNT/A to a BoNT/A receptor, theinternalization of the neurotoxin/receptor complex, the translocation ofthe BoNT/A light chain from an intracellular vesicle into the cytoplasmand the proteolytic cleavage of a SNAP-25. Also referred to as agenetically-engineered cell line, cells from such an established cellline may, e.g., express an exogenous FGFR2, an exogenous FGFR3, anexogenous SV2, an exogenous SNAP-25, or any combination thereof. As usedherein, the term “established clonal cell line” or “clonal cell line”refers to a cell culture of cells selected from a heterogenouspopulation of cell types comprising an established cell line, which canalso be referred to as a parental cell line. Thus, as a non-limitingexample, the cells from the A6, A7, A9, A10, A11, B5, B10, C5, C11, C12,D7, D11, E11, F10, H1, H3, H8, H10, 1E3, 2B9, 2E4, 3B8, 3D5, 3G10, 4B5,4C8, 5F3, AC9, AF4, BB3, BB10, BE3, BF8, CC11, CD6, CE6, CG8, CG10, DC4,DD10, DE7, YF5, and H1 1.4 clonal cell lines were selected from theheterogenous population of cell types comprising the SiMa cell line (orparental SiMa cell line) DSMZ ACC 164.

Aspects of the present disclosure comprise, in part, a cell from anestablished clonal cell line susceptible to BoNT/A intoxication. As usedherein, the terms “cell(s) susceptible to BoNT/A intoxication,” “cell(s)susceptible to BoNT/A intoxication by a BoNT/A,” or “cell(s) from anestablished clonal cell line susceptible to BoNT/A intoxication by aBoNT/A” refer to cell(s) that can undergo the overall cellular mechanismwhereby a BoNT/A proteolytically cleaves a SNAP-25 substrate andencompasses the binding of a BoNT/A to a BoNT/A receptor, theinternalization of the neurotoxin/receptor complex, the translocation ofthe BoNT/A light chain from an intracellular vesicle into the cytoplasmand the proteolytic cleavage of a SNAP-25. By definition, cell(s)susceptible to BoNT/A intoxication must express, or be engineered toexpress, at least one BoNT/A receptor and at least one SNAP-25substrate. Non-limiting examples of a cell or cells from an establishedclonal cell line susceptible to BoNT/A intoxication, include A6, A7, A9,A10, A11, B5, B10, C5, C11, C12, D7, D11, E11, F10, H1, H3, H8, H10,1E3, 2B9, 2D2, 2D6, 2E4, 3B8, 3D5, 3G10, 4B5, 4C8, 4D3, 5C10, 5F3, AC9,AF4, BB3, BB10, BE3, BF8, CC11, CD6, CE6, CG8, CG10, DC4, DD10, DE7,DF5, YB7, YF5, and H1 1.4 clonal cell lines. As used herein, the terms“cell(s) that can uptake BoNT/A” or “cell(s) comprising an establishedclonal cell line that can uptake BoNT/A” refer to cells that can undergothe overall cellular mechanism whereby a BoNT/A proteolytically cleavesa SNAP-25 substrate and encompasses the binding of a BoNT/A to a BoNT/Areceptor, the internalization of the neurotoxin/receptor complex, thetranslocation of the BoNT/A light chain from an intracellular vesicleinto the cytoplasm and the proteolytic cleavage of a SNAP-25. Bydefinition, cell(s) that can uptake BoNT/A must express, or beengineered to express, at least one BoNT/A receptor and at least oneSNAP-25 substrate. Non-limiting examples of a cell or cells from anestablished clonal cell line that can uptake BoNT/A, include A6, A7, A9,A10, A11, B5, B10, C5, C11, C12, D7, D11, E11, F10, H1, H3, H8, H10,1E3, 2B9, 2D2, 2D6, 2E4, 3B8, 3D5, 3G10, 4B5, 4C8, 4D3, 5C10, 5F3, AC9,AF4, BB3, BB10, BE3, BF8, CC11, CD6, CE6, CG8, CG10, DC4, DD10, DE7,DF5, YB7, YF5, and H1 1.4 clonal cell lines. Cell lines comprisingcell(s) from an established clonal cell line susceptible to BoNT/Aintoxication or cell(s) that can uptake BoNT/A are referred to as a“responder cell line.”

Aspects of the present disclosure comprise, in part, cells from anestablished clonal cell line more susceptible to BoNT/A intoxication. Asused herein, the terms “cell(s) more susceptible to BoNT/Aintoxication,” “cell(s) more susceptible to BoNT/A intoxication by aBoNT/A,” or “cell(s) from an established clonal cell line moresusceptible to BoNT/A intoxication by a BoNT/A” refer to cell(s) thatundergo the overall cellular mechanism whereby a BoNT/A proteolyticallycleaves a SNAP-25 substrate to a greater degree as compared to a cell(s)comprising a parental SiMa cell line, such as, e.g., parental SiMa cellline DSMZ ACC 164. Non-limiting examples of a cell or cells from anestablished clonal cell line more susceptible to BoNT/A intoxication,include a cell or cells from a A10, H1, 2E4, 3B8, 3D5, 5F3, AF4, BB3,BB10, DC4, or H1 1.4 clonal cell lines. As used herein, the terms“cell(s) that uptake BoNT/A more” or “cell(s) comprising an establishedclonal cell line that uptake BoNT/A more” refer to cells that undergothe overall cellular mechanism whereby a BoNT/A proteolytically cleavesa SNAP-25 substrate to a greater degree as compared to a cell(s)comprising a parental SiMa cell line, such as, e.g., parental SiMa cellline DSMZ ACC 164. Non-limiting examples of a cell or cells from anestablished clonal cell line that can uptake BoNT/A more, include a cellor cells from a A10, H1, 2E4, 3B8, 3D5, 5F3, AF4, BB3, BB10, DC4, or H11.4 clonal cell lines. Cell lines comprising cell(s) from an establishedclonal cell line more susceptible to BoNT/A intoxication or cell(s) thatcan uptake BoNT/A more are referred to as a “high responder cell line.”

Aspects of the present disclosure comprise, in part, cells from anestablished clonal cell line less susceptible to BoNT/A intoxication. Asused herein, the terms “cell(s) less susceptible to BoNT/Aintoxication,” “cell(s) less susceptible to BoNT/A intoxication by aBoNT/A,” or “cell(s) from an established clonal cell line lesssusceptible to BoNT/A intoxication by a BoNT/A” refer to cell(s) thatundergo the overall cellular mechanism whereby a BoNT/A proteolyticallycleaves a SNAP-25 substrate to a lesser degree as compared to a cell(s)comprising a parental SiMa cell line, such as, e.g., parental SiMa cellline DSMZ ACC 164. Non-limiting examples of a cell or cells from anestablished clonal cell line less susceptible to BoNT/A intoxication,include A7, B10, D11, H10, 2D2, 2D6, 4D3, 5C10, DF5, and YB7 clonal celllines. As used herein, the terms “cell(s) that uptake BoNT/A less” or“cell(s) comprising an established clonal cell line that uptake BoNT/Aless” refer to cells that undergo the overall cellular mechanism wherebya BoNT/A proteolytically cleaves a SNAP-25 substrate to a lesser degreeas compared to a cell(s) comprising a parental SiMa cell line, such as,e.g., parental SiMa cell line DSMZ ACC 164. Non-limiting examples of acell or cells from an established clonal cell line that can uptakeBoNT/A less, include A7, B10, D11, H10, 2D2, 2D6, 4D3, 5C10, DF5, andYB7 clonal cell lines. Cell lines comprising cell(s) from an establishedclonal cell line less susceptible to BoNT/A intoxication or cell(s) thatcan uptake BoNT/A less are referred to as a “low responder cell line.”

Aspects of the present disclosure comprise, in part, a cell from anestablished clonal cell line not susceptible to BoNT/A intoxication. Asused herein, the terms “cell(s) not susceptible to BoNT/A intoxication,”“cell(s) not susceptible to BoNT/A intoxication by a BoNT/A,” or“cell(s) from an established clonal cell line not susceptible to BoNT/Aintoxication by a BoNT/A” refer to cell(s) that cannot undergo theoverall cellular mechanism whereby a BoNT/A proteolytically cleaves aSNAP-25 substrate. As used herein, the terms “cell(s) that cannot uptakeBoNT/A” or “cell(s) comprising an established clonal cell line thatcannot uptake BoNT/A” refer to cells that cannot undergo the overallcellular mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25substrate. Cell lines comprising cell(s) from an established clonal cellline not susceptible to BoNT/A intoxication or cell(s) that cannotuptake BoNT/A are referred to as a “non-responder cell line.”

Thus in an embodiment, cells from an established clonal cell line aresusceptible to BoNT/A intoxication. In aspects of this embodiment, cellsfrom an established clonal cell line are susceptible to BoNT/Aintoxication by, e.g., about 500 pM or less, about 400 pM or less, about300 pM or less, about 200 pM or less, or about 100 pM or less of aBoNT/A. In other aspects of this embodiment, cells from an establishedclonal cell line are susceptible to BoNT/A intoxication by, e.g., about90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM orless, about 50 pM or less, about 40 pM or less, about 30 pM or less,about 20 pM or less, or about 10 pM or less of a BoNT/A. In still otheraspects, cells from an established clonal cell line are susceptible toBoNT/A intoxication by, e.g., about 9 pM or less, about 8 pM or less,about 7 pM or less, about 6 pM or less, about 5 pM or less, about 4 pMor less, about 3 pM or less, about 2 pM or less, or about 1 pM or lessof a BoNT/A. In yet other aspects, cells from an established clonal cellline are susceptible to BoNT/A intoxication by, e.g., about 0.9 pM orless, about 0.8 pM or less, about 0.7 pM or less, about 0.6 pM or less,about 0.5 pM or less, about 0.4 pM or less, about 0.3 pM or less, about0.2 pM, or about 0.1 pM or less of a BoNT/A. As used herein, the term“about” when qualifying a value of a stated item, number, percentage, orterm refers to a range of plus or minus ten percent of the value of thestated item, percentage, parameter, or term.

In another embodiment, cells comprising an established clonal cell linecan uptake a BoNT/A. In aspects of this embodiment, cells comprising anestablished clonal cell line can uptake, e.g., about 500 pM or less,about 400 pM or less, about 300 pM or less, about 200 pM or less, orabout 100 pM or less of a BoNT/A. In other aspects of this embodiment,cells comprising an established clonal cell line possess the ability touptake about 90 pM or less, about 80 pM or less, about 70 pM or less,about 60 pM or less, about 50 pM or less, about 40 pM or less, about 30pM or less, about 20 pM or less, or about 10 pM or less of a BoNT/A. Instill other aspects, cells comprising an established clonal cell linepossess the ability to uptake about 9 pM or less, about 8 pM or less,about 7 pM or less, about 6 pM or less, about 5 pM or less, about 4 pMor less, about 3 pM or less, about 2 pM or less, or about 1 pM or lessof a BoNT/A. In yet other aspects, cells comprising an establishedclonal cell line possess the ability to uptake about 0.9 pM or less,about 0.8 pM or less, about 0.7 pM or less, about 0.6 pM or less, about0.5 pM or less, about 0.4 pM or less, about 0.3 pM or less, about 0.2 pMor less, or about 0.1 pM or less of a BoNT/A. In other aspects, cellscomprising an established clonal cell line can uptake BoNT/A from, e.g.,about 0.01 pM to about 100 pM, about 0.01 pM to about 75 pM, about 0.01pM to about 50 pM, about 0.01 pM to about 25 pM, about 0.01 pM to about20 pM, about 0.01 pM to about 15 pM, about 0.01 pM to about 10 pM, about0.01 pM to about 5 pM, about 0.001 pM to about 100 pM, about 0.001 pM toabout 75 pM, about 0.001 pM to about 50 pM, about 0.001 pM to about 25pM, about 0.001 pM to about 20 pM, about 0.001 pM to about 15 pM, about0.001 pM to about 10 pM, or about 0.001 pM to about 5 pM of BoNT/A.

In another embodiment, cells from an established clonal cell line arenot susceptible to BoNT/A intoxication. In aspects of this embodiment,cells from an established clonal cell line are not susceptible to BoNT/Aintoxication by, e.g., about 500 pM or less, about 400 pM or less, about300 pM or less, about 200 pM or less, or about 100 pM or less of aBoNT/A. In other aspects of this embodiment, cells from an establishedclonal cell line are susceptible to BoNT/A intoxication by, e.g., about90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM orless, about 50 pM or less, about 40 pM or less, about 30 pM or less,about 20 pM or less A, or about 10 pM or less of a BoNT/A. In stillother aspects, cells from an established clonal cell line are notsusceptible to BoNT/A intoxication by, e.g., about 9 pM or less, about 8pM or less, about 7 pM or less, about 6 pM or less, about 5 pM or less,about 4 pM or less, about 3 pM or less, about 2 pM or less, or about 1pM or less of a BoNT/A. In yet other aspects, cells from an establishedclonal cell line are not susceptible to BoNT/A intoxication by, e.g.,about 0.9 pM or less, about 0.8 pM or less, about 0.7 pM or less, about0.6 pM or less, about 0.5 pM or less, about 0.4 pM or less, about 0.3 pMor less, about 0.2 pM, or about 0.1 pM or less of a BoNT/A. In otheraspects, cells from an established clonal cell line are not susceptibleto BoNT/A intoxication from, e.g., about 0.01 pM to about 100 pM, about0.01 pM to about 75 pM, about 0.01 pM to about 50 pM, about 0.01 pM toabout 25 pM, about 0.01 pM to about 20 pM, about 0.01 pM to about 15 pM,about 0.01 pM to about 10 pM, about 0.01 pM to about 5 pM, about 0.001pM to about 100 pM, about 0.001 pM to about 75 pM, about 0.001 pM toabout 50 pM, about 0.001 pM to about 25 pM, about 0.001 pM to about 20pM, about 0.001 pM to about 15 pM, about 0.001 pM to about 10 pM, orabout 0.001 pM to about 5 pM of BoNT/A.

Aspects of the present disclosure comprise, in part, cells from anestablished clonal cell line susceptible to BoNT/A intoxication that aremore stable than cells from a parental SiMa cell line, such as, e.g.,parental SiMa cell line DSMZ ACC 164. As used herein, the term “stable”refers to cells from an established clonal cell line for a particularpassage number that exhibit a relative EC₅₀, sensitivity, efficacy,well-defined upper asymptote, and/or a well-defined dose-response curvefor BoNT/A activity that is similar to the values for relative EC₅₀,sensitivity, efficacy, well-defined upper asymptote, and/or awell-defined dose-response curve exhibited by cells from a parental SiMacell line, such as, e.g., parental SiMa cell line DSMZ ACC 164, at thesame or similar passage number, where the same assay conditions and thesame BoNT/A (or molecule) are used in both assays.

Thus in an embodiment, cells from an established clonal cell line aremore stable as compared to a parental SiMa cell line. In an aspect ofthis embodiment, cells from an established clonal cell line are morestable as compared to the parental SiMa cell line DSMZ ACC 164. In otheraspects of this embodiment, cells from an established clonal cell lineare more stable for, e.g., at least 5 more passages, at least 10 morepassages, at least 15 more passages, at least 20 more passages, at least25 more passages, or at least 30 more passages, as compared to aparental SiMa cell line. In yet other aspects of this embodiment, cellsfrom an established clonal cell line are more stable for, e.g., at least5 more passages, at least 10 more passages, at least 15 more passages,at least 20 more passages, at least 25 more passages, or at least 30more passages, as compared to a parental SiMa cell line DSMZ ACC 164.

Aspects of the present disclosure comprise, in part, cells from anestablished clonal cell line susceptible to BoNT/A intoxication that arestable over a plurality of cell passages. As used herein, the term“stable” refers to cells from an established clonal cell line for aparticular passage number that exhibit a relative EC₅₀, sensitivity,efficacy, well-defined upper asymptote, and/or a well-defineddose-response curve for BoNT/A activity that is similar to the valuesfor relative EC₅₀, sensitivity, efficacy, well-defined upper asymptote,and/or a well-defined dose-response curve exhibited by cells from thesame established clonal cell line, but from a prior passage or passages,where the same assay conditions and the same BoNT/A (or molecule) areused in both assays.

Cells from an established cell line disclosed in the presentspecification can exhibit consistent sensitivity to BoNT/A activity overa plurality of cell passages. As used herein, the term “sensitivity toBoNT/A activity” refers to the lowest dose that an assay can measureconsistently above the signal detected by a non-treatment control orbackground signal.

Thus, in an embodiment, cells from the established clonal cell lineexhibit a sensitivity for BoNT/A activity for any given passages that ise.g., 100 pM or less, about 80 pM or less, about 70 pM or less, about 60pM or less, about 50 pM or less, about 40 pM or less, about 30 pM orless, about 20 pM or less, about 10 pM or less, about 1 pM or less,about 0.9 pM or less, about 0.8 pM or less, about 0.7 pM or less, about0.6 pM or less, about 0.5 pM or less, about 0.4 pM or less, about 0.3 pMor less, about 0.2 pM or less, or about 0.1 pM or less of a BoNT/A. Inaspects of this embodiment, cells from the established clonal cell lineexhibit a sensitivity for BoNT/A activity for any given passages thatis, e.g., about 0.01 pM to about 100 pM, about 0.01 pM to about 75 pM,about 0.01 pM to about 50 pM, about 0.01 pM to about 25 pM, about 0.01pM to about 20 pM, about 0.01 pM to about 15 pM, about 0.01 pM to about10 pM, about 0.01 pM to about 5 pM, about 0.001 pM to about 100 pM,about 0.001 pM to about 75 pM, about 0.001 pM to about 50 pM, about0.001 pM to about 25 pM, about 0.001 pM to about 20 pM, about 0.001 pMto about 15 pM, about 0.001 pM to about 10 pM, or about 0.001 pM toabout 5 pM of BoNT/A.

In another embodiment, cells from the established clonal cell lineexhibit a sensitivity for BoNT/A activity that is about 100 pM or less,about 75 pM or less, about 50 pM or less, about 25 pM or less, lessabout 20 pM or less, about 15 pM or less, about 10 pM or less, or about1 pM or less for, e.g., 5 or more cell passages, 10 or more cellpassages, 15 or more cell passages, 20 or more cell passages, 25 or morecell passages, 30 or more cell passages, 35 or more cell passages, 40 ormore cell passages, 45 or more cell passages, or 50 or more cellpassages. In other aspects of this embodiment, cells from theestablished clonal cell line exhibit a sensitivity for BoNT/A activitythat is about 100 pM or less, about 75 pM or less, about 50 pM or less,about 25 pM or less, less about 20 pM or less, about 15 pM or less,about 10 pM or less, or about 1 pM or less for, e.g., about 15 to about60 passages, about 20 to about 60 passages, about 25 to about 60passages, about 30 to about 60 passages, about 35 to about 60 passages,about 40 to about 60 passages, about 45 to about 60 passages, about 50to about 60 passages, about 15 to about 50 passages, about 20 to about50 passages, about 25 to about 50 passages, about 30 to about 50passages, about 35 to about 50 passages, about 40 to about 50 passages,about 15 to about 40 passages, about 20 to about 40 passages, about 25to about 40 passages, or about 30 to about 40 passages.

Cells from an established cell line disclosed in the presentspecification can exhibit a consistent relative efficacy of BoNT/Auptake, BoNT/A activity, or BoNT/A intoxication over a plurality of cellpassages. As used herein, the term “relative efficacy” refers to howwell the upper asymptote for the BoNT/A activity detected in the currentassay run compares to the upper asymptote for the BoNT/A activitydetected in a reference standard, a reference molecule, or a referencepassage number used on that assay. As used herein, the term “signal tonoise ratio for the upper asymptote” refers to the signal detected in anassay at the upper limit of detection divided by the signal detected bya non-treatment control or background signal. The upper limit ofdetection is the highest dose that an assay can measure consistentlybefore saturation of the signal occurs.

Thus, in an embodiment, cells from an established cell line disclosed inthe present specification can exhibit a well defined upper asymptoteover a plurality of cell passages and maintain a signal to noise ratiothat is consistent and adequate for the assay. In aspects of thisembodiment, cells from an established cell line disclosed in the presentspecification must have a signal to noise ratio for the upper asymptotefor BoNT/A activity of, e.g., at least 3:1, at least 4:1, at least 5:1,at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1,at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least35:1, at least 40:1, at least 45:1, at least 50:1, at least 60:1, atleast 70:1, at least 80:1, at least 90:1, or at least 100:1, at least150:1, at least 200:1, at least 250:1, at least 300:1, at least 350:1,at least 400:1, at least 450:1, at least 500:1, at least 550:1, or atleast 600:1, over, e.g., 5 or more cell passages, 10 or more cellpassages, 15 or more cell passages, 20 or more cell passages, 25 or morecell passages, 30 or more cell passages, 35 or more cell passages, 40 ormore cell passages, 45 or more cell passages, or 50 or more cellpassages. In other aspects of this embodiment, cells from an establishedcell line disclosed in the present specification must have a signal tonoise ratio for the upper asymptote for BoNT/A activity of, e.g., atleast 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, atleast 8:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, atleast 25:1, at least 30:1, at least 35:1, at least 40:1, at least 45:1,at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least90:1, or at least 100:1, at least 150:1, at least 200:1, at least 250:1,at least 300:1, at least 350:1, at least 400:1, at least 450:1, at least500:1, at least 550:1, or at least 600:1, over, e.g., about 15 to about60 passages, about 20 to about 60 passages, about 25 to about 60passages, about 30 to about 60 passages, about 35 to about 60 passages,about 40 to about 60 passages, about 45 to about 60 passages, about 50to about 60 passages, about 15 to about 50 passages, about 20 to about50 passages, about 25 to about 50 passages, about 30 to about 50passages, about 35 to about 50 passages, about 40 to about 50 passages,about 15 to about 40 passages, about 20 to about 40 passages, about 25to about 40 passages, or about 30 to about 40 passages.

Cells from an established cell line disclosed in the presentspecification can exhibit a well defined dose-response curve for BoNT/Aactivity over a plurality of cell passages. As used herein, the term“dose-response curve” refers to the how well the raw data fits thestatistical model of choice for that assay. As a non-limiting example, asigmoidal curve with a four parameter logistics fit is a dose-responsecurve for an enzymatic activity assay, such as, e.g., a potency assay.As another non-limiting example, a ligand binding with one sitesaturation fit is a dose-response curve for a ligand/antibody bindingassay.

Thus, in an embodiment, cells from an established cell line disclosed inthe present specification exhibit a well defined dose-response curve forBoNT/A activity over a plurality of cell passages. In aspects of thisembodiment, cells from an established cell line disclosed in the presentspecification exhibit a well defined dose-response curve for BoNT/Aactivity over, e.g., 5 or more cell passages, 10 or more cell passages,15 or more cell passages, 20 or more cell passages, 25 or more cellpassages, 30 or more cell passages, 35 or more cell passages, 40 or morecell passages, 45 or more cell passages, or 50 or more cell passages. Inother aspects of this embodiment, cells from an established cell linedisclosed in the present specification exhibit a well defineddose-response curve for BoNT/A activity over, e.g., about 15 to about 60passages, about 20 to about 60 passages, about 25 to about 60 passages,about 30 to about 60 passages, about 35 to about 60 passages, about 40to about 60 passages, about 45 to about 60 passages, about 50 to about60 passages, about 15 to about 50 passages, about 20 to about 50passages, about 25 to about 50 passages, about 30 to about 50 passages,about 35 to about 50 passages, about 40 to about 50 passages, about 15to about 40 passages, about 20 to about 40 passages, about 25 to about40 passages, or about 30 to about 40 passages.

Cells from an established cell line disclosed in the presentspecification can exhibit a consistent relative EC₅₀ value for BoNT/Aactivity over a plurality of cell passages. As used herein, the term“relative EC₅₀” or “relative EC₅₀ value” refers to an EC₅₀ value ofBoNT/A activity that is normalized against the EC₅₀ calculated for areference standard, a reference molecule, or a reference passage numberused on that assay.

Thus, in an embodiment, cells from an established clonal cell lineexhibit a consistent relative EC₅₀ for BoNT/A activity over a pluralityof cell passages. In aspects of this embodiment, cells from anestablished clonal cell line exhibit a consistent relative EC₅₀ forBoNT/A activity that is, e.g., about ±10%, about ±20%, about ±30%, about±40%, about ±50%, about 60%, about 70%, or about ±75%, the relative EC₅₀for BoNT/A activity over, e.g., 5 or more cell passages, 10 or more cellpassages, 15 or more cell passages, 20 or more cell passages, 25 or morecell passages, 30 or more cell passages, 35 or more cell passages, 40 ormore cell passages, 45 or more cell passages, or 50 or more cellpassages. In other aspects of this embodiment, cells from an establishedclonal cell line exhibit a relative EC₅₀ for BoNT/A activity that is,e.g., about ±10% to about 75%, about ±10% to about 70%, about ±10% toabout 60%, about ±10% to about 50%, about ±10% to about 40%, about ±10%to about 30%, or about ±10% to about 20% the relative EC₅₀ for BoNT/Aactivity over, e.g., 5 or more cell passages, 10 or more cell passages,15 or more cell passages, 20 or more cell passages, 25 or more cellpassages, 30 or more cell passages, 35 or more cell passages, 40 or morecell passages, 45 or more cell passages, or 50 or more cell passages.

Aspects of the present disclosure comprise, in part, cells from anestablished clonal cell line susceptible to BoNT/A intoxication thatexhibit at least a 1.5-fold difference in gene expression levels of oneor more genes listed in Tables 5, 6, 7, or 8 as compared to theexpression levels of these genes in cells from the 2D6 cell line. Inother aspects of this embodiment, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit a difference in geneexpression levels of one or more genes listed in Tables 5, 6, 7, or 8of, e.g., at least a 1.5-fold, at least a 2.0-fold, at least a 2.5-fold,at least a 3.0-fold, at least a 3.5-fold, at least a 4.0-fold, at leasta 4.5-fold, at least a 5.0-fold, at least a 5.5-fold, at least a6.0-fold, at least a 7.0-fold, or at least a 8.0-fold as compared to theexpression levels of these genes in cells from the 2D6 cell line. Inother aspects of this embodiment, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit a difference in geneexpression levels of, e.g., 2 or more genes, 3 or more genes, 4 or moregenes, 5 or more genes, 6 or more genes, 7 or more genes, 8 or moregenes, 9 or more genes, 10 or more genes, 20 or more genes, 30 or moregenes, 40 or more genes, 50 or more genes, 60 or more genes, 70 or moregenes, 80 or more genes, 90 or more genes, or 100 or more genes listedin Tables 5, 6, 7, or 8 as compared to the expression levels of thesegenes in cells from the 2D6 cell line. In yet other aspects, cells froman established clonal cell line susceptible to BoNT/A intoxicationexhibit a difference in gene expression levels of, e.g., about 5 genesto about 100 genes, about 10 genes to about 100 genes, about 15 genes toabout 100 genes, about 20 genes to about 100 genes, about 25 genes toabout 100 genes, about 5 genes to about 75 genes, about 10 genes toabout 75 genes, about 15 genes to about 75 genes, about 20 genes toabout 75 genes, about 25 genes to about 75 genes, about 5 genes to about50 genes, about 10 genes to about 50 genes, about 15 genes to about 50genes, about 20 genes to about 50 genes, or about 25 genes to about 50genes listed in Tables 5, 6, 7, or 8 as compared to the expressionlevels of these genes in cells from the 2D6 cell line. The log ratio inTables 5, 6, 7, and 8 represent log₂ values where 0.585 is log₂(1.5)which is a 1.5-fold difference, 1 is log₂(2) which is a 2-folddifference, 1.584 is log₂(3) which is a 3-fold difference, 2 is log₂(4)which is a 4-fold difference, 2.321 is log₂(5) which is a 5-folddifference, 2.584 is log₂(6) which is a 6-fold difference, 2.807 islog₂(7) which is a 7-fold difference, 3 is log₂(8) which is a 8-folddifference, 3.169 is log₂(9) which is a 9-fold difference, and 3.321 islog₂(10) which is a 10-fold difference.

In an embodiment, cells from an established clonal cell line susceptibleto BoNT/A intoxication exhibit at least a 1.5-fold increase in geneexpression levels of one or more genes listed in Tables 5 or 7 ascompared to the expression levels of these genes in cells from the 2D6cell line. In aspects of this embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit an increasein gene expression levels of one or more genes listed in Tables 5 or 7of, e.g., at least a 2.0-fold, at least a 2.5-fold, at least a 3.0-fold,at least a 3.5-fold, at least a 4.0-fold, at least a 4.5-fold, at leasta 5.0-fold, at least a 5.5-fold, at least a 6.0-fold, at least a7.0-fold, or at least a 8.0-fold as compared to the expression levels ofthese genes in cells from the 2D6 cell line. In other aspects of thisembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit an increase in gene expression levels of,e.g., 2 or more genes, 3 or more genes, 4 or more genes, 5 or moregenes, 6 or more genes, 7 or more genes, 8 or more genes, 9 or moregenes, 10 or more genes, 20 or more genes, 30 or more genes, 40 or moregenes, 50 or more genes, 60 or more genes, 70 or more genes, 80 or moregenes, 90 or more genes, or 100 or more genes listed in Tables 5 or 7 ascompared to the expression levels of these genes in cells from the 2D6cell line. In yet other aspects, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit an increase in geneexpression levels of, e.g., about 5 genes to about 100 genes, about 10genes to about 100 genes, about 15 genes to about 100 genes, about 20genes to about 100 genes, about 25 genes to about 100 genes, about 5genes to about 75 genes, about 10 genes to about 75 genes, about 15genes to about 75 genes, about 20 genes to about 75 genes, about 25genes to about 75 genes, about 5 genes to about 50 genes, about 10 genesto about 50 genes, about 15 genes to about 50 genes, about 20 genes toabout 50 genes, or about 25 genes to about 50 genes listed in Tables 5or 7 as compared to the expression levels of these genes in cells fromthe 2D6 cell line.

In an embodiment, cells from an established clonal cell line susceptibleto BoNT/A intoxication exhibit at least a 1.5-fold increase in geneexpression levels of one or more genes selected from A2BP1, ACO1, ACOT7,ACOT9, ACTL6A, ACTL6B, ADRBK2, AGPS, AIDA, AKAP13, ALCAM, ALDH7A1,AMOTL1, ANAPC7, ANKRD13A, ANKRD54, ANKS1B, ANLN, ANP32E, ANXA6, APAF1,APBB2, APOO, ARF3, ARHGAP11A, ARHGAP24, ARHGAP24, ARHGAP6, ARHGEF3,ARHGEF6, ARL13B, ARL6, ARL6IP1, ASCL1, ASF1A, ASF1B, ASXL3, ATAD2,ATP6V1A, AURKA, AURKB, BARD1, BASP1, BAX, BCL11A, BCR, BNC2, BOK, BRCA1,BRCA2, BRD4, BRI3BP, BRIP1, BTBD3, BTG3, BUB1, BUB1B, BUB3, BVES,C10ORF58, C10ORF78, C11ORF75, C12ORF48, C12ORF49, C14ORF106, C14ORF143,C15ORF23, C15ORF42, C18ORF54, C1ORF112, C1ORF183, C1ORF43, C1QL1,C20ORF108, C20ORF20, C20ORF7, C20ORF72, C22ORF28, C22ORF29, C22ORF39,C3ORF14, C3ORF70, C4ORF46, C4ORF49, C5ORF32, C6ORF115, C9ORF100,CARHSP1, CBLB, CBWD1, CBX5, CCDC109B, CCDC117, CCDC15, CCDC21, CCDC3,CCDC34, CCDC52, CCDC86, CCDC99, CCNB1, CCNE2, CCNF, CCNYL1, CD24, CD47,CD9, CDC2, CDC25B, CDC42EP4, CDC45L, CDC5L, CDC6, CDCA2, CDCA4, CDCA5,CDCA7, CDCA8, CDH2, CDK2, CDK2AP1, CDK6, CDKAL1, CDKN2C, CDKN2D, CDO1,CDS2, CECR5, CELSR3, CENPF, CENPH), CENPI, CENPJ, CENPK, CENPL, CENPM,CENPN, CENPO, CEP135, CEP152, CEP55, CEP78, CEP97, CHAF1A, CHD6, CHEK1,CHEK2, CHMP4B, CHRNA3, CHRNA7, CHST15, CIT, CKAP2, CKAP2L, CKLF, CKS1B,CLDND1, CLSTN2, CMTM7, CNN3, CNOT4, CNP, CNTN1, CNTN4, COBLL1, COQ3,CPNE4, CPT1A, CPVL, CRISPLD1, CRTAC1, CRYBG3, CRYZ, CSE1L, CSRP2,CSRP2BP, CSTF1, CTDSPL2, CTNNBL1, CTSL2, CUGBP2, CXCR4, CYTSA, DACT1,DAZ1, DBF4B, DBH, DCLRE1B, DCPS, DDAH2, DDT, DEK, DENR, DEPDC1, DEPDC1B,DERA, DGCR14, DGKE, DHX15, DHX35, DIAPH1, DIAPH3, DKFZP434L187, DLEU2,DLGAP5, DLL3, DLX6, DNA2, DNM3, DNMT3B, DOCK10, DOK4, DOK5, DPF1, DPYD,DPYSL3, DRAM1, DRG1, DSCC1, DSN1, DTL, DTNBP1, DTYMK, DVL2, DYNLT3,DYRK4, E2F1, E2F2, E2F7, E2F8, EAF2, EBF1, ECT2, EFNA5, EFNB2, ELAVL4,ELOVL7, EME1, EMILIN2, EMILIN3, EML1, ENC1, EPB41L5, EPOR, ERCC6L,ESCO2, ESF1, ESPL1, ETS1, ETV1, EXO1, EXO05, EXOSC6, EXPH5, EZH2,FAM101B, FAM105A, FAM110A, FAM114A1, FAM118A, FAM120C, FAM129B, FAM13A,FAM162B, FAM181B, FAM19A4, FAM19A5, FAM54A, FAM64A, FAM7A3, FAM83D,FANCG, FANCI, FANCL, FANCM, FARP1, FAT1, FBLN1, FBN1, FBP1, FBXO43,FBXO5, FEN1, FGFR2, FH, FIGNL1, FKBP5, FNDC5, FOXD1, FOXN3, FRMD6, FRZB,FSTL1, FZD2, FZD5, FZD8, G2E3, GABBR2, GAP43, GAP43, GAS2L3, GATM,GEMIN4, GFPT2, GFRA1, GFRA2, GGCX, GINS1, GINS3, GJC1, GLDC, GMNN,GNAI1, GNASAS, GNB4, GNG11, GNG12, GNG4, GPAM, GPN3, GPR125, GPR161,GPSM2, GPX3, GRP, GSG2, GSS, GSTCD, GTSE1, GTSF1, GXYLT1, H1F0, H2AFX,HAT1, HAUS6, HDAC8, HEG1, HES6, HGF, HIC2, HMG4L, HMGB1, HMGB2, HMGB3,HMGXB4, HMMR, HOOK3, HPS4, HRH3, HS3ST2, HS6ST2, HSPB11, HTATSF1, IDH2,IFI27L1, IQGAP1, ITGA6, ITGAV, ITGB5, ITPRIP, JARID2, KCNG3, KCNJ8,KCNN1, KCTD12, KDELC2, KHDRBS3, KIAA0101, KIAA0406, KIAA1211, KIAA1524,KIF11, KIF14, KIF16B, KIF18A, KIF23, KIFC1, KLF7, KLHL13, KLHL5, KNTC1,KPNA2, LBH, LGR5, LHFPL2, LIFR, LIG3, LMF2, LMNB1, LOC100127983,LOC100128844, LOC100288551, LOC340109, LOC344595, LOC440288, LOC642597,LOC728052, LPAR1, LRFN2, LRRC1, LSM4, LUM, MAB21L1, MAB21L2, MAGEH1,MAN1A1, MAN2A1, MAOA, MAP3K13, MAPK11, MAPK12, MAPKAPK3, MASTL, MBD2,MCAM, MCM10, MCM3, MCM4, MCM5, MCM6, MCM7, MCM8, MDC1, MDM1, MED20,MEIS1, MEIS2, MELK, MEST, MFAP2, MGAT5B, MINA, MKI67, MKL1, MLF11P, MMD,MNS1, MPHOSPH9, MPP5, MPPED2, MRC2, MRPL35, MRPL49, MRS2, MSN, MTF2,MTHFD1, MTUS1, MVK, MYBL1, MYBL2, MYD88, MYO1B, MYO6, MYST1, NAAA, NAGA,NANP, NARG1, NASP, NAT11, NAT13, NCAPD3, NCAPG, NCAPG2, NCAPH, NCAPH2,NDN, NEDD1, NEDD9, NEIL3, NELL2, NFIB, NOC4L, NOS1, NR3C1, NRG3, NRM,NRXN1, NSMCE4A, NTAN1, NUCKS1, NUDT1, NUF2, NUP107, NUP37, NUP43, NUP50,NUP93, NUSAP1, NXPH1, NXT2, ODZ3, ODZ4, OIP5, ORC1L, ORC6L, OSBPL3,PAICS, PANK2, PARP3, PASK, PBK, PCDH17, PCDH8, PCGF5, PCNA, PDCL, PDE5A,PDLIM5, PDRG1, PEG3, PELI2, PELO, PEX13, PEX26, PFKFB3, PGAM5, PGM2,PHF19, PHF20, PHF21B, PHF5A, PHLPP1, PHYHIPL, PI4KA, PIP4K2A, PIR,PKMYT1, PLCL2, PLD5, PLEKHF2, PLK2, PLK4, PLS3, PLXNA2, PLXNA4, PLXNB2,PM20D2, POLA1, POLA2, POLE, POLE2, POLQ, POSTN, PPAP2B, PPAT, PPIF,PPIL5, PPM1F, PPP1R13B, PPP1R3C, PPP2R2B, PPP3CB, PRIM1, PRIM2, PRLHR,PRPS1, PRSS12, PSMA7, PSMC3IP, PSMD5, PSMD9, PSRC1, PTER, PTGES2,PTGFRN, PTGR1, PTPRE, PTPRG, PTPRK, PTPRM, PTTG1, PVRL3, PXMP4, PXN,RAB35, RAB3B, RAD18, RAD51, RAD51C, RALY, RAN, RANBP1, RANGAP1, RASEF,RBBP7, RBBP9, RBL1, RBPMS, RBX1, RECQL4, REEP1, RELB, RELL1, REXO2,RFC1, RFC2, RFC3, RFC4, RFC5, RGMA, RGS5, RILPL2, RIMBP2, RMI1,RNASEH2A, RNF182, RNF26, RNF34, RPA1, RPP30, RRM1, RRM2, RRP7A, RSRC1,S1PR3, SALL4, SAP30, SASS6, SBF2, SCD5, SCML1, SCML2, SDC1, SEC23A,SEC61A2, SELM, SEMA6A, SEMA6D, SESN3, SEZ6L, SFI1, SFRP1, SGOL2, SH3D19,SHCBP1, SHMT1, SHOX2, SIM1, SIVA1, SKA1, SKA2, SKA3, SKP2, SLC25A10,SLC26A2, SLC2A4RG, SLC2A8, SLC43A3, SLC44A5, SLC7A2, SLC8A1, SLCO3A1,SLIT3, SLITRK5, SLK, SLMO2, SMC1A, SMC2, SMC4, SMC6, SMO, SMYD5, SNAI2,SNAP29, SNCAIP, SNRPD3, SNX18, SNX5, SORD, SOX4, SPAG6, SPARC, SPATS2L,SPC24, SPC25, SPOCK1, SPSB4, SRGAP1, SRRD, SRRM4, SSH2, ST8SIA1,ST8SIA2, ST8SIA4, STARD4, STIL, STK17A, STMN3, STOM, STON2, STRA6,SUSD5, SUV39H1, SVIP, SYN2, SYNE2, SYNM, SYT17, SYTL4, TBC1D1, TBX3,TCF19, TCF7L2, TCFL5, TDRKH, TEAD3, TESC, TFDP1, TFDP2, TFPI2, TH1L,THOC4, TIFA, TIMP3, TLE3, TLE4, TMCC3, TMEM107, TMEM132C, TMEM151B,TMEM170B, TMEM178, TMEM194A, TMEM38B, TMEM48, TMEM56, TMEM98, TMPO,TMSB15B, TNFAIP8, TOB2, TOMM34, TPBG, TPTE, TPX2, TRIM29, TRIM36,TRIM68, TRIM9, TRIP13, TROAP, TSHZ3, TSPAN14, TSPAN4, TSPAN5, TTC28,TTC9, TUBA1B, TUBB, TWIST1, TXNRD1, TYMS, TYRO3, UBE2C, UBE2L3, UBE2V1,UBE3B, UCK1, UCP2, UFD1L, UHRF1, UNC119B, UNG, UQCC, USP1, USP48, UTP18,VAPB, VAV3, VPS29, VSTM2L, WDHD1, WDR51A, WDR53, WDR62, WDR67, WEE1,WEE1, WHSC1, WRAP53, WWC3, XRCC4, XRCC6, ZAK, ZFHX4, ZFP82, ZGPAT,ZNF215, ZNF217, ZNF238, ZNF253, ZNF280B, ZNF367, ZNF43, ZNF443, ZNF503,ZNF521, ZNF560, ZNF608, ZNF626, ZNF681, ZNF71, ZNF823, ZNF85, ZNF92,ZNF93, and/or ZWINT, as compared to the expression levels of these genesin cells from the 2D6 cell line.

In an aspect of this embodiment, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit at least a 1.5-foldincrease in gene expression levels of one or more genes selected fromA2BP1, ACOT9, ALCAM, ANLN, ARHGAP24, ARHGAP6, ARHGEF3, ARHGEF6, ASCL1,ASF1B, ATAD2, AURKA, AURKB, BARD1, BASP1, BNC2, BRCA2, BRIP1, BTG3,BUB1, BUB1B, BVES, C11ORF75, C12ORF48, C14ORF106, C15ORF42, C18ORF54,C1ORF112, C3ORF70, C4ORF49, CCDC109B, CCDC3, CCNB1, CCNE2, CD9, CDC2,CDC25B, CDC45L, CDC6, CDCA2, CDCA5, CDCA8, CDK2, CDKN2D, CENPF, CENPI,CENPL, CENPN, CEP55, CHEK1, CHEK2, CHRNA7, CIT, CKAP2L, CLSTN2, CNTN1,CNTN4, CPVL, CRYBG3, CSRP2, CTSL2, CUGBP2, CXCR4, DAZ1, DEPDC1, DEPDC1B,DIAPH3, DLGAP5, DNA2, DOK5, DPYD, DPYSL3, DSCC1, DSN1, DTL, DYNLT3,E2F1, E2F2, E2F7, E2F8, ECT2, EFNB2, ELOVL7, EME1, EMILIN2, EMILIN3,EML1, ENC1, ERCC6L, ESCO2, ESPL1, ETV1, EXO1, EXPH5, FAM101B, FAM114A1,FAM54A, FAM64A, FAM7A3, FAM83D, FANCI, FAT1, FBLN1, FBP1, FBXO43, FGFR2,FNDC5, FOXD1, FRMD6, FRZB, FZD5, GAS2L3, GFRA2, GINS1, GINS3, GNAI1,GNB4, GNG11, GNG12, GPSM2, GRP, GTSE1, GTSF1, HGF, HMMR, HS3ST2, ITGA6,ITPRIP, KCNG3, KCTD12, KDELC2, KIAA0101, KIAA1524, KIF11, KIF14, KIF18A,KIF23, KIFC1, LOC340109, LOC642597, LOC728052, LOC728052, LPAR1, LUM,MAB21L1, MAB21L2, MAOA, MCM10, MCM5, MELK, MINA, MKI67, MLF11P, MPPED2,MRC2, MSN, MYBL1, MYBL2, MYO6, NCAPG, NCAPH, NDN, NEDD9, NEIL3, NR3C1,NRXN1, NUF2, NUSAP1, OIP5, ORC1L, OSBPL3, PBK, PCGF5, PDE5A, PEG3, PELO,PFKFB3, PHLPP1, PLD5, PLK2, PLK4, PLS3, POLA2, POLE2, POLQ, PPAP2B,PPP1R3C, PRLHR, PRSS12, PSRC1, PTGR1, PTPRE, PTPRK, PTPRM, PTTG1, PVRL3,RAD51, RBL1, RBPMS, RELL1, RFC5, RGMA, RGS5, RNF182, RRM2, S1PR3, SGOL2,SHCBP1, SHOX2, SIM1, SKA1, SLC43A3, SLC44A5, SLC7A2, SLITRK5, SMC2,SMC6, SPAG6, SPARC, SPC24, SPC25, ST8SIA4, STK17A, SUSD5, SYN2, SYNM,SYT17, TCF19, TESC, TFDP2, TFPI2, TIMP3, TLE3, TMEM132C, TMEM178,TNFAIP8, TPBG, TPTE, TPX2, TRIM29, TRIM36, TRIM68, TROAP, TWIST1, TYMS,UBE2C, UBE3B, UHRF1, WDHD1, WDR51A, WDR67, WEE1, ZFP82, ZNF367, ZNF521,and/or ZWINT, as compared to the expression levels of these genes incells from the 2D6 cell line.

In another aspect of this embodiment, cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit at least a 1.5-foldincrease in gene expression levels of one or more genes selected fromA2BP1, ALCAM, ANLN, ARHGAP24, ARHGAP6, ASCL1, ASF1B, ATAD2, AURKA,AURKB, BARD1, BASP1, BNC2, BRIP1, BUB1, BVES, C11ORF75, C12ORF48,C14ORF106, C18ORF54, C3ORF70, CCDC109B, CCDC3, CD9, CDC2, CDC45L, CDCA2,CDCA8, CDK2, CENPL, CEP55, CHEK2, CHRNA7, CKAP2L, CNTN1, CRYBG3, CUGBP2,CXCR4, DAZ1, DEPDC1, DIAPH3, DLGAP5, DOK5, DPYD, DTL, DYNLT3, E2F1,E2F7, E2F8, EFNB2, ELOVL7, EMILIN2, EML1, ENC1, ESPL1, ETV1, EXO1,EXPH5, FAM54A, FAM64A, FAM7A3, FAM83D, FANCI, FAT1, FBLN1, FBXO43,FGFR2, FZD5, GNAI1, GNB4, GNG11, GNG12, GPSM2, GRP, GTSE1, GTSF1, HGF,HMMR, ITGA6, ITPRIP, KCTD12, KDELC2, KIF11, KIF14, KIF18A, KIF23, KIFC1,LOC340109, LOC728052, LPAR1, LUM, MAB21L1, MAB21L2, MAOA, MCM10, MELK,MINA, MKI67, MSN, MYBL1, NCAPH, NDN, NEDD9, NEIL3, NRXN1, NUF2, NUSAP1,OIP5, ORC1L, OSBPL3, PBK, PCGF5, PEG3, PEG3, PHLPP1, PLD5, PLK2, PLK4,PLS3, POLE2, PPAP2B, PPP1R3C, PRLHR, PRSS12, PTPRE, PTPRK, PTPRM, RBPMS,RELL1, RGS5, RNF182, RRM2, S1PR3, SGOL2, SHCBP1, SHOX2, SIM1, SLC43A3,SLC44A5, SLC7A2, SLITRK5, SMC6, SPAG6, SPARC, SPC24, SPC25, ST8SIA4,STK17A, SYT17, TFPI2, TIMP3, TMEM132C, TMEM178, TPBG, TPTE, TPX2,TRIM36, TWIST1, UBE3B, and/or ZNF367, as compared to the expressionlevels of these genes in cells from the 2D6 cell line.

In yet another aspect of this embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit at least a1.5-fold increase in gene expression levels of one or more genesselected from A2BP1, ARHGAP24, ARHGAP6, ASCL1, ASF1B, ATAD2, AURKA,BARD1, BASP1, BNC2, BUB1, BVES, C11ORF75, CCDC3, CHEK2, CHRNA7, CKAP2L,CNTN1, CRYBG3, CUGBP2, CUGBP2, CXCR4, DAZ1, DEPDC1, DIAPH3, DLGAP5,DOK5, DPYD, DTL, DYNLT3, E2F8, EFNB2, ELOVL7, ENC1, EXPH5, FAM7A3,FANCI, FAT1, FBLN1, FGFR2, GNAI1, GNB4, GNG11, GNG12, GRP, GTSE1, GTSF1,HMMR, ITGA6, ITPRIP, KCTD12, KDELC2, LOC728052, LPAR1, LUM, MAB21L1,MAB21L2, MAOA, MCM10, MELK, MINA, MKI67, MSN, MYBL1, NDN, NEDD9, NRXN1,OSBPL3, PEG3, PLK2, PLS3, PRLHR, PRSS12, PTPRK, PTPRM, RBPMS, RGS5,RNF182, RRM2, SGOL2, SIM1, SLC43A3, SLC44A5, SLC7A2, SMC6, SPAG6, SPARC,STK17A, TFPI2, TIMP3, TMEM132C, TMEM178, and/or TPTE, as compared to theexpression levels of these genes in cells from the 2D6 cell line.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from ADAMTS9,ATAD2, C11ORF82, CDC45L, CNTN1, CNTN4, Cyclin A, Cyclin E, E2F1, E2F2,E2F7, ELOVL7, EME1, FGFR, FGFR2, KIAA1524, MELK, MYBL1, MYBL2, NDC80,NDN, ORC1L, PLS3, PRIMA1, RAD54L, RBL1, RBPMS, RRM2, S1PR3, SCLY,SLCIA3, SPC24, SPC25, ST8SIA4, TFDP1, TFP12, TK1, TMEM35, TTK, TWIST1,TYMS, TYK, and/or ZWINT, as compared to the expression levels of thesegenes in cells from the 2D6 cell line.

In yet another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from AURKB,BIRC5, BRCA1, BRCA2, BRIP1, BUB1B, CD9, DLGAP3, DYNLT3, ENC1, FBLN1,FOXM1, Gβγ, GNAI1, GNG11, GNG12, GPSM2, GUCY1B3, HGF, ITGA6, JNK, KCNJ5,KIF18A, KITLG, MMD, MSN, MYRIP, NEK2, NR3Cl, NXPH1, OSBPL3, PKMYT1,PTPRM, RAD51, RAD51AP1, SLC7A2, SLC43A3, SMC6, SNAI2, SNCAIP, SSH2,STK17A, SYNPO2, TOP2A, TPTE, TRAF4, TSPAN, TSPAN4, UBE3, UBE3B, and/orVAV3, as compared to the expression levels of these genes in cells fromthe 2D6 cell line. In still another embodiment, cells from anestablished clonal cell line susceptible to BoNT/A intoxication exhibitat least a 1.5-fold increase in gene expression levels of one or moregenes selected from ALCAM, AURKA, CHEK, CIT, CSRP2, E2F, ECT2, EFNB2,ERK, ESPL1, GNAI, GPR161, HMMR, KIF4A, KIF14, KIF15, KIF22, KIF23,KIFC1, LPAR1, MK167, OIP5, PHLPP, PP1/PP2A, PPP1R3C, PRC1, PTTG1,RACGAP, RB, RGS5, SDC2, and/or TPX2, as compared to the expressionlevels of these genes in cells from the 2D6 cell line.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from ASCL1,HES6, MAPK, NMU, PEG3, PTPRK, PRLHR, PTPRK, SGOL2, SPARC, and/or ZNF217,as compared to the expression levels of these genes in cells from the2D6 cell line. In yet another embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit at least a1.5-fold increase in gene expression levels of one or more genesselected from ASF1B, BASP1, CHAF1A, NCAPH, PBK, PRAME, SMC2, UHRF1,and/or VRK1, as compared to the expression levels of these genes incells from the 2D6 cell line. In still another embodiment, cells from anestablished clonal cell line susceptible to BoNT/A intoxication exhibitat least a 1.5-fold increase in gene expression levels of one or moregenes selected from C14ORF106, CEP72, KIF20A, PCNA, PEX13, PFCS, POLQ,SPAGS, SYTL4, TROAP, and/or WDR51A, as compared to the expression levelsof these genes in cells from the 2D6 cell line. In a further embodiment,cells from an established clonal cell line susceptible to BoNT/Aintoxication exhibit at least a 1.5-fold increase in gene expressionlevels of one or more genes selected from ANLN, ARHGAP24, ASPM, BUB1,CCDC99, CEP55, CKAP2, DRAM, E2F8, PLXNA2, SLC16A10, UBE2C, UBE2S, and/orWDHD1, as compared to the expression levels of these genes in cells fromthe 2D6 cell line.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from ARHGEF3,CTSL2, DIAPH3, FBP1, KIF2C, KIF11, PFKFB3, and/or PLK4, as compared tothe expression levels of these genes in cells from the 2D6 cell line. Inyet another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from ARHGEF3,CTSL2, DIAPH3, FBP1, KIF2C, KIF11, PFKFB3, and/or PLK4, as compared tothe expression levels of these genes in cells from the 2D6 cell line. Instill another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from FNDC5,HSPC159, MAB21L2, SLITRK5, SYN2, and/or ZNF367, as compared to theexpression levels of these genes in cells from the 2D6 cell line. In afurther another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from EXO1,KCTD12, MYO6, PHEBL1, SHCBP1, TPBG, and/or TUBB6, as compared to theexpression levels of these genes in cells from the 2D6 cell line. In yetanother embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from BTG3,GABRA5, TR1P10, and/or ZNF521, as compared to the expression levels ofthese genes in cells from the 2D6 cell line. In still anotherembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit at least a 1.5-fold increase in geneexpression levels of one or more genes selected from BNC2, DPYD,EMILIN2, PPIL5, and/or TACC3, as compared to the expression levels ofthese genes in cells from the 2D6 cell line.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-folddifference in gene expression levels of one or more genes listed inTables 14 or 16 as compared to the expression levels of these genes incells from the 2D6 cell line. In an aspect of this embodiment, cellsfrom an established clonal cell line susceptible to BoNT/A intoxicationexhibit at least a 1.5-fold increase in gene expression levels of one ormore genes selected from ACOT9, ADAMTS9, ARHGAP24, ARHGEF3, ASCL1,BARD1, BASP1, C3ORF70, C11ORF75, CCDC109B, CD9, CDCA7L, CDK2, CENPL,CLSTN2, CNTN1, CSRP2, CTSL2, CUGBP2, DEPDC1, DIAPH3, DOK5, DPYD, DYNLT3,EMILIN2, ETV1, FAM101B, FBLN1, FGFR2, FNDC5, GNAI1, GNB4, GNG11, GNG12,GPR177, GTSE1, HGF, KITLG, LPAR1, MAB21L2, MAOA, MCM10, MINA, MSN, MYO6,MYRIP, PAG1, PEG3, PLK2, POLA2, PPP1R3C, PRLHR, PRSS12, PTGR1, PTPRK,PVRL3, RAB32, RBPMS, SDC2, SGOL2, SLC43A3, SLC7A2, SMC2, SMC6, SPARC,SPC25, ST8SIA4, TCF7L1, TFPI2, TMEM35, TMEM178, TNFAIP8, TPTE, TRIP10,TWIST1, and/or ZNF521, as compared to the expression levels of thesegenes in cells from the 2D6 cell line. In another aspect of thisembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit at least a 1.5-fold increase in geneexpression levels of one or more genes selected from ADAMTS9, ARHGAP24,ASCL1, BARD1, BASP1, BVES, C11ORF75, CDCA7L, CNTN1, CUGBP2, DOK5, DPYD,DYNLT3, FBLN1, FGFR2, GNAI1, GNB4, GNG11, GNG12, GTSE1, GTSF1, ITPRIP,KDELC2, LOC728052, LPAR1, MAB21L2, MAOA, MINA, MSN, PEG3, PLK2, PRLHR,PRSS12, PTPRK, RBPMS, RNF182, SGOL2, SLC43A3, SLC44A5, SLC7A2, SMC6,SPARC, TFPI2, TMEM178, and/or TPTE, as compared to the expression levelsof these genes in cells from the 2D6 cell line. In yet another aspect ofthis embodiment, cells from an established clonal cell line susceptibleto BoNT/A intoxication exhibit at least a 1.5-fold increase in geneexpression levels of one or more genes selected from ADAMTS9, ASCL1,BASP1, DOK5, DPYD, GNB4, GNG11, GTSF1, MAOA, MINA, MSN, PEG3, PLK2,PRSS12, RNF182, SLC44A5, SPARC, TFPI2, and/or TPTE, as compared to theexpression levels of these genes in cells from the 2D6 cell line.

In yet another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from ADAMTS9,ARHGAP24, ARHGEF3, ASCL1, BARD1, CD9, CDK2, CSRP2, CTSL2, DIAPH3, DOK5,DYNLT3, EMILIN2, ETV1, FBLN1, FGFR2, GNAI1, GNB4, GNG11, GNG12, HGF,KITLG, LPAR1, MCM10, MSN, PAG1, PEG3, PLK2, POLA2, PPP1R3C, PTPRK,RAB32, SDC2, SLC43A3, SLC7A2, SMC6, SPARC, SPC25, ST8SIA4, TCF7L1,TFPI2, TMEM35, TNFAIP8, TPTE, TRIP10, and/or TWIST1, as compared to theexpression levels of these genes in cells from the 2D6 cell line. In anaspect of this embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from ADAMTS9,ARHGAP24, ASCL1, BARD1, DOK5, DYNLT3, FBLN1, FGFR2, GNAI1, GNB4, GNG11,GNG12, LPAR1, MSN, PEG3, PLK2, PTPRK, SLC43A3, SLC7A2, SMC6, SPARC,TFPI2, and/or TPTE, as compared to the expression levels of these genesin cells from the 2D6 cell line. In another aspect of this embodiment,cells from an established clonal cell line susceptible to BoNT/Aintoxication exhibit at least a 1.5-fold increase in gene expressionlevels of one or more genes selected from ADAMTS9, ASCL1, DOK5, GNB4,GNG11, MSN, PEG3, PLK2, SPARC, TFPI2, and/or TPTE, as compared to theexpression levels of these genes in cells from the 2D6 cell line.

In still another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from ACOT9,BASP1, C11ORF75, CCDC109B, CDCA7L, CLSTN2, CNTN1, CUGBP2, DEPDC1, DPYD,FAM101B, FNDC5, GTSE1, MAOA, MINA, MYO6, MYRIP, PLK2, PRLHR, PVRL3,RBPMS, SGOL2, SMC2, TFP12, TMEM178, and/or ZNF521, as compared to theexpression levels of these genes in cells from the 2D6 cell line. In anaspect of this embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from BASP1,C11ORF75, CDCA7L, CNTN1, CUGBP2, DPYD, GTSE1, MAOA, MINA, PLK2, PRLHR,RBPMS, SGOL2, TFPI2, and/or TMEM178, as compared to the expressionlevels of these genes in cells from the 2D6 cell line. In another aspectof this embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from BASP1,DPYD, MAOA, MINA, PLK2, and/or TFPI2, as compared to the expressionlevels of these genes in cells from the 2D6 cell line.

In a further embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from C3ORF70,MAB21L2, PRSS12, CENPL, GPR177, and/or PTGR1, as compared to theexpression levels of these genes in cells from the 2D6 cell line. In anaspect of this embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from MAB21L2and/or PRSS12, as compared to the expression levels of these genes incells from the 2D6 cell line.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold decreasein gene expression levels of one or more genes listed in Tables 6 or 8as compared to the expression levels of these genes in cells from a 2D6cell line. In other aspects of this embodiment, cells from anestablished clonal cell line susceptible to BoNT/A intoxication exhibita decrease in gene expression levels of one or more genes listed inTables 6 or 8 of, e.g., at least a 1.5-fold, at least a 2.0-fold, atleast a 2.5-fold, at least a 3.0-fold, at least a 3.5-fold, at least a4.0-fold, at least a 4.5-fold, at least a 5.0-fold, at least a 5.5-fold,at least a 6.0-fold, at least a 7.0-fold, or at least a 8.0-fold ascompared to the expression levels of these genes in cells from a 2D6cell line. In other aspects of this embodiment, cells from anestablished clonal cell line susceptible to BoNT/A intoxication exhibita decrease in gene expression levels of, e.g., 2 or more genes, 3 ormore genes, 4 or more genes, 5 or more genes, 6 or more genes, 7 or moregenes, 8 or more genes, 9 or more genes, 10 or more genes, 20 or moregenes, 30 or more genes, 40 or more genes, 50 or more genes, 60 or moregenes, 70 or more genes, 80 or more genes, 90 or more genes, or 100 ormore genes listed in Tables 6 or 8 as compared to the expression levelsof these genes in cells from the 2D6 cell line. In yet other aspects ofthis embodiment, cells from an established clonal cell line susceptibleto BoNT/A intoxication exhibit an decrease in gene expression levels of,e.g., about 5 genes to about 100 genes, about 10 genes to about 100genes, about 15 genes to about 100 genes, about 20 genes to about 100genes, about 25 genes to about 100 genes, about 5 genes to about 75genes, about 10 genes to about 75 genes, about 15 genes to about 75genes, about 20 genes to about 75 genes, about 25 genes to about 75genes, about 5 genes to about 50 genes, about 10 genes to about 50genes, about 15 genes to about 50 genes, about 20 genes to about 50genes, or about 25 genes to about 50 genes listed in Tables 6 or 8 ascompared to the expression levels of these genes in cells from the 2D6cell line.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold decreasein gene expression levels of one or more genes selected from AATK,ABCA2, ABCC5, ABCC8, ABR, ABTB1, ACOT1, ACRBP, ACRV1, ACYP2, ADARB1,ADCY1, ADM, AFF1, AGAP4, AGPAT3, AGXT2L2, AHI1, AKAP9, AKIRIN1, AKT2,AMH, ANK2, ANK3, ANKHD1, ANKRD17, ANKRD50, ANKS1A, AQP1, ARF1, ARHGAP1,ARHGAP23, ARL17A, ASAM, ASPHD1, ATP2B3, ATP2B3, ATP6V0A1, ATP8B2, ATP9A,BACE1, BACH2, BLZF1, BPTF, BRUNOL4, BRUNOL5, BRWD1, BRWD2, BTBD18,BTN3A3, BZRAP1, C14ORF159, C15ORF24, C15ORF57, C16ORF52, C17ORF28,C1ORF21, C1ORF21, C1ORF50, C21ORF57, C21ORF59, C21ORF66, C2CD2, C2CD4A,C2ORF60, C2ORF68, C3ORF23, C4ORF41, C5ORF42, C6ORF154, C6ORF52,C7ORF28B, C7ORF54, C9ORF150, C9ORF68, CACNA1D, CALY, CAMK2B, CAMK2N2,CAP2, CAPN2, CARTPT, CBS, CCDC104, CCDC50, CCDC76, CD151, CD163L1,CD302, CDC42EP1, CDH10, CDH12, CDKN1C, CDKN2A, CDS1, CFC1, CHD5, CLCN3,CLDN12, CLIP4, CLMN, CNGA3, CNNM1, CNOT6L, COL6A1, COPA, CPNE8, CRKRS,CRTC1, CRYGS, CSTB, CTSK, CUL4A, CYGB, CYLD, CYP2E1, CYP3A5, CYTL1,D4S234E, DCLK1, DCTD, DEPDC6, DHX36, DIP2A, DIS3, DKFZP547G183, DNAJC22,DTNA, DTWD1, DUSP16, DUSP5, DVL3, EFNB3, EIF3C, ELFN2, ENOSF1, ERAP1,ERBB2, ERBB4, ERMAP, ETFDH, EXOSC6, FADS3, FAM120AOS, FAM150B, FAM165B,FAM184B, FAM30A, FAM46A, FAM66C, FAT4, FBRSL1, FBXW7, FGD5, FGF13,FLJ33630, FNBP1, FOXO6, FUBP3, GAL, GAL, GLT25D2, GNAS, GOLGA8A, GPR123,GPX7, GRIA2, GRIP2, GRM5, GUCY1A3, H2AFY, HCG 1776018, HCG 2022304,HCN4, HEATR1, HELQ, HERC4, HERPUD2, HEXDC, HGSNAT, HIST1H2AC, HIST1H2AE,HIST1H2BD, HIST1H2BK, HIST1H3I, HIST2H2BE, HIVEP3, HNRNPM, HNRNPR,HPCAL1, HPCAL4, HSPD1, IER3, IL10RB, IL17B, INTU, JMY, KAT2B, KATNB1,KBTBD11, KCNMA1, KCNQ2, KCNS2, KCTD13, KHDC1, KIAA0125, KIAA1370,KIAA1598, KIF1A, KIF5C, KIF5C, KISS1R, KLHDC1, LCORL, LGALS3BP,LOC100130097, LOC100130360, LOC100130522, LOC100272228, LOC284408,LOC399491, LOC401320, LOC641298, LOC642852, LOC90110, LOC94431, LONRF2,LRP2BP, LRRC37A2, LRRFIP1, LTBP3, LYPLAL1, LYRM5, MAP3K5, MAP7, MAP9,MCM3AP, MCM3APAS, MCTP1, MEOX2, METTL3, MFSD4, MGAT4A, MLXIP, MRPL1,MRPS33, MST1, MUC20, MXD1, MZF1, NAMPT, NAP1L3, NBPF1, NBPF10, NCOA7,NCRNA00171, NCRNA00182, NDRG1, NDUFA4L2, NDUFV3, NEBL, NELF, NHEDC2,NHEG1, NIPAL2, NIPAL3, NLRX1, NOL3, NSMAF, NUDT19, NUP153, NUP54, OLFM3,OR7D2, OSBP, PABPC1L, PABPC4, PACRGL, PAM, PAPPA, PAQR6, PARP12, PCBD2,PCNXL2, PDCD6, PDE4C, PDE9A, PDGFRB, PDIA2, PGAP1, PHF17, PHKA1, PHKA2,PHLDA2, PIGH, PION, PKD1, PLA2G4C, PLCB4, PLEKHH1, PLP2, PLRG1, PLXNC1,PNCK, PNMA3, POFUT2, POGK, PPAPDC1A, PPAPDC1B, PPID, PPIE, PPM1K,PPP1R2, PPP2R2C, PPT1, PRKACB, PRPH, PSMB7, PTCD1, PTGER2, PTGS1, PTN,PTPRD, PTPRN, RAB11FIP3, RAB6B, RAF1, RGAG4, RGS11, RGS8, RHBDL1, RHOQ,RHOU, RLF, RNASET2, RNF13, RNF149, RNF165, RNF207, RNF41, RPL37, RSL1D1,RUNDC3A, S100A6, SCMH1, SCN2A, SERP1, SETX, SFRS18, SFXN3, SGK3, SH3BP5,SH3GL2, SH3YL1, SHC2, SHC4, SIGIRR, SIK1, SLC12A7, SLC1A2, SLC1A6,SLC22A17, SLC35F3, SLC38A5, SLCO1A2, SMAGP, SMAP2, SNCA, SOBP, SORCS1,SORL1, SPINT2, SPIRE2, SPOCK2, SRR, ST8SIA3, STAR, STEAP3, STOX2, STX3,SYNJ1, SYT13, SYT5, TAF10, TANC2, TCEA1, THBS2, THSD4, TIMP1, TM2D1,TMEM111, TMEM151A, TMEM184C, TMEM41B, TMEM43, TMEM5, TMEM59L, TMIE,TNFRSF25, TNFRSF25, TOX4, TP53BP1, TPM3, TRA2A, TRIM33, TRIM73, TRIMS,TRIT1, TSR1, TTC17, TTC3, TTC39C, TUSC3, TXLNB, U2AF1, UBE2D3, UBE2G2,UBN2, UBXN6, UCN, UGP2, UNC80, UNQ1887, USP36, USPL1, VN1R1, VPS13C,VPS53, VPS8, WDFY3, WDR27, WDR85, WSB1, YJEFN3, YLPM1, ZDHHC11, ZER1,ZG16B, ZNF275, ZNF440, ZNF573, ZNF641, ZNF662, ZNF785, and/or ZNF814, ascompared to the expression levels of these genes in cells from the 2D6cell line.

In an aspect of this embodiment, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit at least a 1.5-folddecrease in gene expression levels of one or more genes selected fromACOT1, ADARB1, ADM, ANK2, AQP1, ASAM, C2CD4A, C3ORF23, CACNA1D, CAMK2B,CAP2, CARTPT, CD163L1, CDC42EP1, CDH10, CDKN1C, CDKN2A, CFC1, CLMN,CPNE8, CYGB, CYP2E1, CYTL1, DNAJC22, DUSP5, ENOSF1, ERAP1, FGF13, FOXO6,GAL, GNAS, GPX7, GRIA2, GRM5, HCG 1776018, H1ST1H2AC, HIST1H2BD,HIST1H2BK, HIST2H2BE, IL17B, KIAA0125, KIAA1598, KISS1R, LRP2BP, MEOX2,NCRNA00182, NDRG1, NDUFA4L2, NIPAL2, NUDT19, PDE4C, PHKA2, PHLDA2,PTGER2, RGS11, S100A6, SCN2A, SHC4, SIGIRR, SLC1A2, SLC1A6, SLC35F3,SLC38A5, SORCS1, SYT13, SYT5, THBS2, TIMP1, and/or TMEM111, as comparedto the expression levels of these genes in cells from the 2D6 cell line.

In another aspect of this embodiment, cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit at least a 1.5-folddecrease in gene expression levels of one or more genes selected fromACOT1, ADARB1, ANK2, AQP1, C3ORF23, CAP2, CARTPT, CD163L1, CDC42EP1,CDKN2A, CFC1, CPNE8, CYGB, CYP2E1, CYTL1, FGF13, FOXO6, GNAS, GRIA2,GRM5, H1ST1H2AC, IL17B, KIAA0125, KIAA1598, KISS1R, MEOX2, NDRG1,NDUFA4L2, NIPAL2, PDE4C, PHKA2, RGS11, SCN2A, SLC1A2, SLC35F3, SLC38A5,SYT13, and/or THBS2, as compared to the expression levels of these genesin cells from the 2D6 cell line.

In yet another aspect of this embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit at least a1.5-fold decrease in gene expression levels of one or more genesselected from AQP1, CAP2, CARTPT, CD163L1, CDC42EP1, CFC1, CPNE8, CYGB,CYP2E1, CYTL1, FGF13, GNAS, GRIA2, GRM5, H1ST1H2AC, IL17B, KIAA0125,MEOX2, NDUFA4L2, PDE4C, PHKA2, RGS11, SLC1A2, SLC35F3, SLC38A5, and/orSYT13, as compared to the expression levels of these genes in cells fromthe 2D6 cell line.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold decreasein gene expression levels of one or more genes selected from ANXA2,AQP1, ARHGAP9, CDH10, CDKN2A, CHPT1, CNTN2, ERAP1, and/or RGS11, ascompared to the expression levels of these genes in cells from the 2D6cell line. In yet another embodiment, cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit at least a 1.5-folddecrease in gene expression levels of one or more genes selected fromABCC8, AELIM3, CAP2, IL17B, MEF2A, NEEBL, PHC, S100A6, SLC1A6, SMAD1,SMAD5, SMAD8, SYT13, and/or SYTL1, as compared to the expression levelsof these genes in cells from the 2D6 cell line. In still anotherembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit at least a 1.5-fold decrease in geneexpression levels of one or more genes selected from CSTB, GPCR, GRIM5,KISSR, SCN2A, SLC1A2, and/or THBS2, as compared to the expression levelsof these genes in cells from the 2D6 cell line.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold decreasein gene expression levels of one or more genes selected from ADARB1,ADM, PTPPH, and/or SLCO1A2, as compared to the expression levels ofthese genes in cells from the 2D6 cell line. In yet another embodiment,cells from an established clonal cell line susceptible to BoNT/Aintoxication exhibit at least a 1.5-fold decrease in gene expressionlevels of one or more genes selected from CNGA3, HIST1H3E, and/or PTGS1,as compared to the expression levels of these genes in cells from the2D6 cell line. In still another embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit at least a1.5-fold decrease in gene expression levels of one or more genesselected from HIST1H2BD and/or OSCAR as compared to the expressionlevels of these genes in cells from the 2D6 cell line. In a furtherembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit at least a 1.5-fold decrease in geneexpression levels of one or more genes selected from CALY as compared tothe expression levels of these genes in cells from the 2D6 cell line.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold decreasein gene expression levels of one or more genes selected from COL5A1and/or MICAL2 as compared to the expression levels of these genes incells from the 2D6 cell line. In yet another embodiment, cells from anestablished clonal cell line susceptible to BoNT/A intoxication exhibitat least a 1.5-fold decrease in gene expression levels of one or moregenes selected from OLFML2A and/or SIGIRR as compared to the expressionlevels of these genes in cells from the 2D6 cell line. In still anotherembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit at least a 1.5-fold decrease in geneexpression levels of one or more genes selected from HPCAL1 and/or LPAR5as compared to the expression levels of these genes in cells from the2D6 cell line. In a further embodiment, cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit at least a 1.5-folddecrease in gene expression levels of one or more genes selected fromHTR1E and/or SORCS1 as compared to the expression levels of these genesin cells from the 2D6 cell line.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold decreasein gene expression levels of one or more genes selected from ANK2,CPNE8, CREB5, IL17B, KIAA0125, LOC100289109, LOC144571, NPAS4, SLC1A2,SORCS1, THBS2, and/or ZNF814, as compared to the expression levels ofthese genes in cells from the 2D6 cell line.

Aspects of the present disclosure comprise, in part, cells from anestablished clonal cell line susceptible to BoNT/A intoxication thatexhibit at least a 1.5-fold difference in gene expression levels of oneor more genes listed in Tables 9, 10, 11, or 12 as compared to theexpression levels of these genes in cells from a parental SiMa cellline. In aspects of this embodiment, cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit at least a 1.5-folddifference in gene expression levels of one or more genes listed inTables 9, 10, 11, or 12 as compared to the expression levels of thesegenes in cells from the parental SiMa cell line DSMZ ACC 164. In otheraspects of this embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit a difference in geneexpression levels of one or more genes listed in Tables 9, 10, 11, or 12of, e.g., at least a 1.5-fold, at least a 2.0-fold, at least a 2.5-fold,at least a 3.0-fold, at least a 3.5-fold, at least a 4.0-fold, at leasta 4.5-fold, at least a 5.0-fold, at least a 5.5-fold, at least a6.0-fold, at least a 7.0-fold, or at least a 8.0-fold as compared to theexpression levels of these genes in cells from the parental SiMa cellline DSMZ ACC 164. In yet other aspects of this embodiment, cells froman established clonal cell line susceptible to BoNT/A intoxicationexhibit a difference in gene expression levels of, e.g., 2 or moregenes, 3 or more genes, 4 or more genes, 5 or more genes, 6 or moregenes, 7 or more genes, 8 or more genes, 9 or more genes, 10 or moregenes, 20 or more genes, 30 or more genes, 40 or more genes, 50 or moregenes, 60 or more genes, 70 or more genes, 80 or more genes, 90 or moregenes, or 100 or more genes listed in Tables 9, 10, 11, or 12 ascompared to the expression levels of these genes in cells from theparental SiMa cell line DSMZ ACC 164. In still other aspects of thisembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit a difference in gene expression levels of,e.g., about 5 genes to about 100 genes, about 10 genes to about 100genes, about 15 genes to about 100 genes, about 20 genes to about 100genes, about 25 genes to about 100 genes, about 5 genes to about 75genes, about 10 genes to about 75 genes, about 15 genes to about 75genes, about 20 genes to about 75 genes, about 25 genes to about 75genes, about 5 genes to about 50 genes, about 10 genes to about 50genes, about 15 genes to about 50 genes, about 20 genes to about 50genes, or about 25 genes to about 50 genes listed in Tables 9, 10, 11,or 12 as compared to the expression levels of these genes in cells fromthe parental SiMa cell line DSMZ ACC 164. The log ratio in Tables 9, 10,11, or 12 represent log₂ values where 0.585 is log₂(1.5) which is a1.5-fold difference, 1 is log₂(2) which is a 2-fold difference, 1.584 islog₂(3) which is a 3-fold difference, 2 is log₂(4) which is a 4-folddifference, 2.321 is log₂(5) which is a 5-fold difference, 2.584 islog₂(6) which is a 6-fold difference, 2.807 is log₂(7) which is a 7-folddifference, 3 is log₂(8) which is a 8-fold difference, 3.169 is log₂(9)which is a 9-fold difference, and 3.321 is log₂(10) which is a 10-folddifference.

In an embodiment, cells from an established clonal cell line susceptibleto BoNT/A intoxication exhibit at least a 1.5-fold difference in geneexpression levels of one or more genes selected from ACOT9, ADAMTS9,ARHGAP24, ARHGEF3, ASCL1, BARD1, BASP1, C3ORF70, C11ORF75, CCDC109B,CD9, CDCA7L, CDK2, CENPL, CLSTN2, CNTN1, CSRP2, CTSL2, CUGBP2, DEPDC1,DIAPH3, DOK5, DPYD, DYNLT3, EMILIN2, ETV1, FAM101B, FBLN1, FGFR2, FNDC5,GNAI1, GNB4, GNG11, GNG12, GPR177, GTSE1, HGF, KITLG, LPAR1, MAB21L2,MAOA, MCM10, MINA, MSN, MYO6, MYRIP, PAG1, PEG3, PLK2, POLA2, PPP1R3C,PRLHR, PRSS12, PTGR1, PTPRK, PVRL3, RAB32, RBPMS, SDC2, SGOL2, SLC43A3,SLC7A2, SMC2, SMC6, SPARC, SPC25, ST8SIA4, TCF7L1, TFPI2, TMEM35,TMEM178, TNFAIP8, TPTE, TRIP10, TWIST1, and/or ZNF521, as compared tothe expression levels of these genes in cells from the parental SiMacell line DSMZ ACC 164. In another aspect of this embodiment, cells froman established clonal cell line susceptible to BoNT/A intoxicationexhibit at least a 1.5-fold difference in gene expression levels of oneor more genes selected from ADAMTS9, ARHGAP24, ASCL1, BARD1, BASP1,BVES, C11ORF75, CDCA7L, CNTN1, CUGBP2, DOK5, DPYD, DYNLT3, FBLN1, FGFR2,GNAI1, GNB4, GNG11, GNG12, GTSE1, GTSF1, ITPRIP, KDELC2, LOC728052,LPAR1, MAB21L2, MAOA, MINA, MSN, PEG3, PLK2, PRLHR, PRSS12, PTPRK,RBPMS, RNF182, SGOL2, SLC43A3, SLC44A5, SLC7A2, SMC6, SPARC, TFPI2,TMEM178, and/or TPTE, as compared to the expression levels of thesegenes in cells from the parental SiMa cell line DSMZ ACC 164. In yetanother aspect of this embodiment, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit at least a 1.5-folddifference in gene expression levels of one or more genes selected fromADAMTS9, ASCL1, BASP1, DOK5, DPYD, GNB4, GNG11, GTSF1, MAOA, MINA, MSN,PEG3, PLK2, PRSS12, RNF182, SLC44A5, SPARC, TFPI2, and/or TPTE, ascompared to the expression levels of these genes in cells from theparental SiMa cell line DSMZ ACC 164.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes listed in Tables 9 or 11as compared to the expression levels of these genes in cells from aparental SiMa cell line DSMZ ACC 164. In other aspects of thisembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit an increase in gene expression levels of oneor more genes listed in Tables 9 or 11 of, e.g., at least a 1.5-fold, atleast a 2.0-fold, at least a 2.5-fold, at least a 3.0-fold, at least a3.5-fold, at least a 4.0-fold, at least a 4.5-fold, at least a 5.0-fold,at least a 5.5-fold, at least a 6.0-fold, at least a 7.0-fold, or atleast a 8.0-fold as compared to the expression levels of these genes incells from a parental SiMa cell line DSMZ ACC 164. In other aspects ofthis embodiment, cells from an established clonal cell line susceptibleto BoNT/A intoxication exhibit an increase in gene expression levels of,e.g., 2 or more genes, 3 or more genes, 4 or more genes, 5 or moregenes, 6 or more genes, 7 or more genes, 8 or more genes, 9 or moregenes, 10 or more genes, 20 or more genes, 30 or more genes, 40 or moregenes, 50 or more genes, 60 or more genes, 70 or more genes, 80 or moregenes, 90 or more genes, or 100 or more genes listed in Tables 9 or 11as compared to the expression levels of these genes in cells from theparental SiMa cell line DSMZ ACC 164. In yet other aspects of thisembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit an increase in gene expression levels of,e.g., about 5 genes to about 100 genes, about 10 genes to about 100genes, about 15 genes to about 100 genes, about 20 genes to about 100genes, about 25 genes to about 100 genes, about 5 genes to about 75genes, about 10 genes to about 75 genes, about 15 genes to about 75genes, about 20 genes to about 75 genes, about 25 genes to about 75genes, about 5 genes to about 50 genes, about 10 genes to about 50genes, about 15 genes to about 50 genes, about 20 genes to about 50genes, or about 25 genes to about 50 genes listed in Tables 9 or 11 ascompared to the expression levels of these genes in cells from theparental SiMa cell line DSMZ ACC 164.

In an embodiment, cells from an established clonal cell line susceptibleto BoNT/A intoxication exhibit at least a 1.5-fold increase in geneexpression levels of one or more genes selected from A2BP1, ADRM1, AFF4,AGAP4, ALCAM, ANAPC7, ANKRD13D, ANKRD28, API5, ARF1, ARHGAP6, ARHGEF10,ARHGEF6, ARL17A, ASCL1, ASXL1, ASXL3, ATF71P, B3GALNT2, BASP1, BLCAP,BNC2, C10ORF58, C12ORF49, C12ORF51, C1ORF43, C20ORF117, C20ORF7, CADM1,CADPS, CALCB, CAPN1, CAPRIN2, CBLB, CBLN2, CBWD1, CCDC150, CCDC3, CD9,CDC25B, CFDP1, CHRNA7, CIAPIN1, CLASP2, CNOT7, CPVL, CRYBG3, CSE1L,CTTNBP2NL, CUGBP2, CXCR4, DACT1, DAZ1, DBH, DCAF8, DENR, DHX35,DKFZP434L187, DLEU2, DLL1, DLL3, DYNC2H1, EAF2, EFNA5, EIF3B, EIF3C,ELOVL7, EML1, EML4, ENC1, EVL, EXOSC6, EXPH5, EZH2, FAM178A, FAM181B,FAM19A4, FAM7A3, FLJ10213, FUBP3, FUS, FZD5, GAP43, GFM1, GFRA2, GGA2,GNAI1, GNAS, GNB4, GNG11, GOLGA4, GPR125, GRM8, GRP, GSPT1, GSS, GSTCD,GULP1, HAUS2, HEG1, HNRNPL, HNRNPM, HOXA7, HOXD4, ID2, IDH3B, IL1A,INSM1, IREB2, ITGA6, ITGB5, KCTD12, KDELC2, KHDRBS3, KIAA0907, KIAA1267,KIF16B, KLC1, KLHL13, LBH, LMO4, LOC100128844, LOC340109, LOC641298,LOC647190, LOC728052, LOC728153, LRPPRC, LRRFIP2, LSM3, LUM, MAB21L1,MAB21L2, MAN2A1, MAOA, MARS, MDN1, MED13L, MED22, MGC24103, MINA, MKLN1,MLEC, MMD, MORF4L2, MPZL1, MSH6, MSI2, MSN, MTMR9, MYO1B, MYO6, N4BP2,NAAA, NDUFS8, NEDD9, NFIB, NKTR, NLN, NOS1, NR2Cl, NUFIP2, NUPL1,OSBPL3, PAPD4, PCBP2, PCDH9, PCGF5, PCNX, PDLIM5, PDZRN3, PELI2, PFKFB3,PGP, PHF20, PLK2, PLXNA2, PLXNA4, PM20D2, POLE, POLQ, PPP2R2A, PPP2R3C,PRDX2, PSMA7, PSME4, PTGFRN, PTK2, PTPN1, PTPRE, PTPRG, QKI, RAB12,RAB35, RAB3GAP2, RAD23B, RAF1, RALGAPA1, RAN, RASEF, RBL1, RDH11, RELL1,REPS1, RGS5, RIMBP2, RNF182, RNF34, RNPEP, ROBO2, RPAIN, RPL35A, RPRD1A,S1PR3, SALL4, SBNO1, SCARB2, SDHA, SEMA6A, SEZ6L, SF1, SFRS8, SIM1,SIRT2, SKIL, SLC20A1, SLC44A5, SLC7A2, SLCO3A1, SLITRK5, SMAD4, SMARCC2,SMC3, SNRPB2, SNRPN, SNX5, SOX2, SPAG6, SPAG9, SPATS2L, SPON1, SR140,SSBP2, ST8SIA1, STMN3, STRA6, SYNCRIP, SYTL3, TAF15, TCF7L2, TDG, TERT,TFPI2, TGFBR1, TH1L, THOC4, TIMP3, TLE3, TMEM132C, TMEM178, TMEM181,TNRC6A, TPBG, TRA2A, TRIM29, TRIM36, TSHZ3, TXNRD1, U2AF1, UBE2O,UBE2V1, UBE2Z, UBXN2A, UCHL1, USP25, USP32, USP34, VPS29, XPO1, XPO7,ZFAND6, ZFHX4, ZFYVE16, ZGPAT, ZNF217, ZNF451, ZNF503, and/or ZNF664, ascompared to the expression levels of these genes in cells from theparental SiMa cell line DSMZ ACC 164.

In an aspect of this embodiment, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit at least a 1.5-foldincrease in gene expression levels of one or more genes selected fromALCAM, ARHGAP6, ARHGEF6, ASCL1, BNC2, CBLN2, CCDC150, CCDC3, CHRNA7,CRYBG3, CUGBP2, CXCR4, DAZ1, DKFZP434L187, DLL1, EFNA5, ELOVL7, EML1,EML4, ENC1, EXPH5, FAM7A3, FZD5, GFRA2, GNAI1, GNB4, GNG11, GRP, ID2,ITGA6, KCTD12, LOC100128844, LOC340109, LOC728052, LUM, MAB21L1,MAB21L2, MAOA, MGC24103, MSN, NEDD9, NFIB, OSBPL3, PAPD4, PCDH9, PDZRN3,PLK2, POLQ, PTGFRN, PTPRE, PTPRG, RAD23B, RGS5, RNF182, ROBO2, SIM1,SLC20A1, SLC44A5, SLITRK5, SPAG6, SPAG9, SR140, TFPI2, TIMP3, TMEM132C,TPBG, TRIM29, TRIM36, and/or ZFAND6, as compared to the expressionlevels of these genes in cells from the parental SiMa cell line DSMZ ACC164.

In another aspect of this embodiment, cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit at least a 1.5-foldincrease in gene expression levels of one or more genes selected fromALCAM, ARHGAP6, ARHGEF6, CBLN2, CCDC3, CHRNA7, CRYBG3, CUGBP2, CXCR4,DAZ1, ELOVL7, EML1, EXPH5, FAM7A3, GNB4, GNG11, GRP, ITGA6, KCTD12,LOC340109, LUM, MAB21L1, MAB21L2, MGC24103, PCDH9, PLK2, POLQ, PTPRE,RGS5, RNF182, ROBO2, SIM1, SLC44A5, SLITRK5, SPAG6, TIMP3, and/orTMEM132C, as compared to the expression levels of these genes in cellsfrom the parental SiMa cell line DSMZ ACC 164.

In yet another aspect of this embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit at least a1.5-fold increase in gene expression levels of one or more genesselected from CBLN2, CCDC3, CHRNA7, CRYBG3, CXCR4, DAZ1, EXPH5, FAM7A3,GNB4, GRP, KCTD12, LUM, MGC24103, PCDH9, PLK2, POLQ, PTPRE, RGS5, ROBO2,SIM1, SLITRK5, TIMP3, and/or TMEM132C, as compared to the expressionlevels of these genes in cells from the parental SiMa cell line DSMZ ACC164.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes listed in Tables 14 or 16as compared to the expression levels of these genes in cells from theparental SiMa cell line DSMZ ACC 164. In aspects of this embodiment,cells from an established clonal cell line susceptible to BoNT/Aintoxication exhibit at least a 1.5-fold increase in gene expressionlevels of one or more genes listed in Tables 14 or 16 as compared to theexpression levels of these genes in cells from a parental SiMa cell lineDSMZ ACC 164. In other aspects of this embodiment, cells from anestablished clonal cell line susceptible to BoNT/A intoxication exhibitan increase in gene expression levels of one or more genes listed inTables 14 or 16 of, e.g., at least a 1.5-fold, at least a 2.0-fold, atleast a 2.5-fold, at least a 3.0-fold, at least a 3.5-fold, at least a4.0-fold, at least a 4.5-fold, at least a 5.0-fold, at least a 5.5-fold,at least a 6.0-fold, at least a 7.0-fold, or at least a 8.0-fold ascompared to the expression levels of these genes in cells from aparental SiMa cell line DSMZ ACC 164. In other aspects of thisembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit an increase in gene expression levels of,e.g., 2 or more genes, 3 or more genes, 4 or more genes, 5 or moregenes, 6 or more genes, 7 or more genes, 8 or more genes, 9 or moregenes, 10 or more genes, 20 or more genes, 30 or more genes, 40 or moregenes, 50 or more genes, 60 or more genes, 70 or more genes, 80 or moregenes, 90 or more genes, or 100 or more genes listed in Tables 14 or 16as compared to the expression levels of these genes in cells from theparental SiMa cell line DSMZ ACC 164. In yet other aspects of thisembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit an increase in gene expression levels of,e.g., about 5 genes to about 100 genes, about 10 genes to about 100genes, about 15 genes to about 100 genes, about 20 genes to about 100genes, about 25 genes to about 100 genes, about 5 genes to about 75genes, about 10 genes to about 75 genes, about 15 genes to about 75genes, about 20 genes to about 75 genes, about 25 genes to about 75genes, about 5 genes to about 50 genes, about 10 genes to about 50genes, about 15 genes to about 50 genes, about 20 genes to about 50genes, or about 25 genes to about 50 genes listed in Tables 14 or 16 ascompared to the expression levels of these genes in cells from theparental SiMa cell line DSMZ ACC 164.

In aspects of this embodiment, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit at least a 1.5-foldincrease in gene expression levels of one or more genes selected fromACOT9, ADAMTS9, ARHGAP24, ARHGEF3, ASCL1, BARD1, BASP1, C3ORF70,C11ORF75, CCDC109B, CD9, CDCA7L, CDK2, CENPL, CLSTN2, CNTN1, CSRP2,CTSL2, CUGBP2, DEPDC1, DIAPH3, DOK5, DPYD, DYNLT3, EMILIN2, ETV1,FAM101B, FBLN1, FGFR2, FNDC5, GNAI1, GNB4, GNG11, GNG12, GPR177, GTSE1,HGF, KITLG, LPAR1, MAB21L2, MAOA, MCM10, MINA, MSN, MYO6, MYRIP, PAG1,PEG3, PLK2, POLA2, PPP1R3C, PRLHR, PRSS12, PTGR1, PTPRK, PVRL3, RAB32,RBPMS, SDC2, SGOL2, SLC43A3, SLC7A2, SMC2, SMC6, SPARC, SPC25, ST8SIA4,TCF7L1, TFPI2, TMEM35, TMEM178, TNFAIP8, TPTE, TRIP10, TWIST1, and/orZNF521, as compared to the expression levels of these genes in cellsfrom the parental SiMa cell line DSMZ ACC 164. In another aspect of thisembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit at least a 1.5-fold increase in geneexpression levels of one or more genes selected from ADAMTS9, ARHGAP24,ASCL1, BARD1, BASP1, BVES, C11ORF75, CDCA7L, CNTN1, CUGBP2, DOK5, DPYD,DYNLT3, FBLN1, FGFR2, GNAI1, GNB4, GNG11, GNG12, GTSE1, GTSF1, ITPRIP,KDELC2, LOC728052, LPAR1, MAB21L2, MAOA, MINA, MSN, PEG3, PLK2, PRLHR,PRSS12, PTPRK, RBPMS, RNF182, SGOL2, SLC43A3, SLC44A5, SLC7A2, SMC6,SPARC, TFPI2, TMEM178, and/or TPTE, as compared to the expression levelsof these genes in cells from the parental SiMa cell line DSMZ ACC 164.In yet another aspect of this embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit at least a1.5-fold increase in gene expression levels of one or more genesselected from ADAMTS9, ASCL1, BASP1, DOK5, DPYD, GNB4, GNG11, GTSF1,MAOA, MINA, MSN, PEG3, PLK2, PRSS12, RNF182, SLC44A5, SPARC, TFPI2,and/or TPTE, as compared to the expression levels of these genes incells from the parental SiMa cell line DSMZ ACC 164.

In yet another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from ADAMTS9,ARHGAP24, ARHGEF3, ASCL1, BARD1, CD9, CDK2, CSRP2, CTSL2, DIAPH3, DOK5,DYNLT3, EMILIN2, ETV1, FBLN1, FGFR2, GNAI1, GNB4, GNG11, GNG12, HGF,KITLG, LPAR1, MCM10, MSN, PAG1, PEG3, PLK2, POLA2, PPP1R3C, PTPRK,RAB32, SDC2, SLC43A3, SLC7A2, SMC6, SPARC, SPC25, ST8SIA4, TCF7L1,TFPI2, TMEM35, TNFAIP8, TPTE, TRIP10, and/or TWIST1, as compared to theexpression levels of these genes in cells from the parental SiMa cellline DSMZ ACC 164. In an aspect of this embodiment, cells from anestablished clonal cell line susceptible to BoNT/A intoxication exhibitat least a 1.5-fold increase in gene expression levels of one or moregenes selected from ADAMTS9, ARHGAP24, ASCL1, BARD1, DOK5, DYNLT3,FBLN1, FGFR2, GNAI1, GNB4, GNG11, GNG12, LPAR1, MSN, PEG3, PLK2, PTPRK,SLC43A3, SLC7A2, SMC6, SPARC, TFPI2, and/or TPTE, as compared to theexpression levels of these genes in cells from the parental SiMa cellline DSMZ ACC 164. In another aspect of this embodiment, cells from anestablished clonal cell line susceptible to BoNT/A intoxication exhibitat least a 1.5-fold increase in gene expression levels of one or moregenes selected from ADAMTS9, ASCL1, DOK5, GNB4, GNG11, MSN, PEG3, PLK2,SPARC, TFPI2, and/or TPTE, as compared to the expression levels of thesegenes in cells from the parental SiMa cell line DSMZ ACC 164.

In still another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from ACOT9,BASP1, C11ORF75, CCDC109B, CDCA7L, CLSTN2, CNTN1, CUGBP2, DEPDC1, DPYD,FAM101B, FNDC5, GTSE1, MAOA, MINA, MYO6, MYRIP, PLK2, PRLHR, PVRL3,RBPMS, SGOL2, SMC2, TFP12, TMEM178, and/or ZNF521, as compared to theexpression level of these genes in cells from the parental SiMa cellline DSMZ ACC 164. In an aspect of this embodiment, cells from anestablished clonal cell line susceptible to BoNT/A intoxication exhibitat least a 1.5-fold increase in gene expression levels of one or moregenes selected from BASP1, C11ORF75, CDCA7L, CNTN1, CUGBP2, DPYD, GTSE1,MAOA, MINA, PLK2, PRLHR, RBPMS, SGOL2, TFPI2, and/or TMEM178, ascompared to the expression levels of these genes in cells from theparental SiMa cell line DSMZ ACC 164. In another aspect of thisembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit at least a 1.5-fold increase in geneexpression levels of one or more genes selected from BASP1, DPYD, MAOA,MINA, PLK2, and/or TFPI2, as compared to the expression levels of thesegenes in cells from the parental SiMa cell line DSMZ ACC 164.

In a further embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from C3ORF70,MAB21L2, PRSS12, CENPL, GPR177, and/or PTGR1, as compared to theexpression levels of these genes in cells from the parental SiMa cellline DSMZ ACC 164. In an aspect of this embodiment, cells from anestablished clonal cell line susceptible to BoNT/A intoxication exhibitat least a 1.5-fold increase in gene expression levels of one or moregenes selected from MAB21L2 and/or PRSS12, as compared to the expressionlevels of these genes in cells from the parental SiMa cell line DSMZ ACC164.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold decreasein gene expression levels of one or more genes listed in Tables 10 or 12as compared to the expression levels of these genes in cells from aparental SiMa cell line. In aspects of this embodiment, cells from anestablished clonal cell line susceptible to BoNT/A intoxication exhibitat least a 1.5-fold decrease in gene expression levels of one or moregenes listed in Tables 10 or 12 as compared to the expression levels ofthese genes in cells from the parental SiMa cell line DSMZ ACC 164. Inother aspects of this embodiment, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit a decrease in geneexpression levels of one or more genes listed in Tables 10 or 12 of,e.g., at least a 1.5-fold, at least a 2.0-fold, at least a 2.5-fold, atleast a 3.0-fold, at least a 3.5-fold, at least a 4.0-fold, at least a4.5-fold, at least a 5.0-fold, at least a 5.5-fold, at least a 6.0-fold,at least a 7.0-fold, or at least a 8.0-fold as compared to theexpression levels of these genes in cells from the parental SiMa cellline DSMZ ACC 164. In other aspects cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit a decrease in geneexpression levels of, e.g., 2 or more genes, 3 or more genes, 4 or moregenes, 5 or more genes, 6 or more genes, 7 or more genes, 8 or moregenes, 9 or more genes, 10 or more genes, 20 or more genes, 30 or moregenes, 40 or more genes, 50 or more genes, 60 or more genes, 70 or moregenes, 80 or more genes, 90 or more genes, or 100 or more genes listedin Tables 10 or 12 as compared to the expression levels of these genesin cells from the parental SiMa cell line DSMZ ACC 164. In yet otheraspects cells from an established clonal cell line susceptible to BoNT/Aintoxication exhibit an decrease in gene expression levels of, e.g.,about 5 genes to about 100 genes, about 10 genes to about 100 genes,about 15 genes to about 100 genes, about 20 genes to about 100 genes,about 25 genes to about 100 genes, about 5 genes to about 75 genes,about 10 genes to about 75 genes, about 15 genes to about 75 genes,about 20 genes to about 75 genes, about 25 genes to about 75 genes,about 5 genes to about 50 genes, about 10 genes to about 50 genes, about15 genes to about 50 genes, about 20 genes to about 50 genes, or about25 genes to about 50 genes listed in Tables 10 or 12 as compared to theexpression levels of these genes in cells from the parental SiMa cellline DSMZ ACC 164.

In an embodiment, cells from an established clonal cell line susceptibleto BoNT/A intoxication exhibit at least a 1.5-fold decrease in geneexpression levels of one or more genes selected from 1-Mar, ABCG1,ABTB1, ACOT1, ACSL1, ACVR1, ACVR2A, ACYP2, ADAMTS1, ADAMTS3, ADAMTSL1,ADARB1, ADCY1, ADD2, ADO, AFF3, AKAP12, ALDH1A2, ALKBH1, ALLC, AMN1,ANKH, ANKRA2, ANKRD50, ANXA5, AP3B2, ARMC10, ARMCX5, ASAM, ASB13,ASPHD1, ATG5, ATL3, ATP2B3, ATP2C1, ATRNL1, B3GALTL, BACE1, BACE2,BBS10, BBS7, BCAP29, BCL2, BCORL1, BRUNOL4, BRUNOL6, BRWD1, BTN3A3,C10ORF10, C10ORF104, C11ORF57, C11ORF70, C14ORF167, C15ORF39, C16ORF52,C17ORF69, C18ORF18, C1GALT1C1, C1ORF21, C1ORF25, C1ORF97, C1 RL,C21ORF57, C2CD2, C2CD4A, C2ORF67, C2ORF68, C3ORF23, C4ORF12, C4ORF39,C4ORF41, C5ORF42, C6ORF120, C9ORF150, CADM2, CAMK2D, CAMK2N1, CAMK2N2,CAP2, CAPN2, CARTPT, CCDC126, CCDC40, CCDC50, CCNY, CD248, CD302,CDC2L6, CDC37L1, CDC42EP3, CDH12, CDKN1C, CDKN2A, CDS1, CFC1, CHMP1B,CLCN3, CLDN12, CLMN, CLN8, CNGA3, CNNM1, CNOT6L, COL27A1, COL5A1,COL6A1, COL6A2, COL6A3, COX18, COX5B, CRYGD, CRYZL1, CTPS2, CTSC,CYB5R2, CYGB, CYLD, CYP2E1, CYP2U1, CYTL1, CYYR1, D4S234E, DAB1, DBT,DCAF10, DCLK1, DCTD, DDAH1, DHRSX, DISP1, DKFZP43410714, DKK1, DNAJC12,DNAJC24, DNASE1L1, DNER, DOPEY2, DRAM2, DSCAM, DSCR3, DTX3L, DUSP16,DUSP22, ECEL1, ECHDC3, EEF1D, EEF2K, EFEMP2, EFNB3, EMID1, ENOX2, ERAP1,ERBB2, ERBB4, ETFDH, ETS2, EYA1, FAM13C, FAM162B, FAM165B, FAM172A,FAM175A, FAM190A, FAM26F, FAM46A, FAM49A, FAM71E1, FAM76A, FAM85A,FBXL5, FBXW7, FCRLB, FGD5, FGF1, FGF13, FGF19, FGF3, FGF7, FIP1L1, FKTN,FLJ10038, FLJ35220, FLJ35390, FLJ37798, FLJ39051, FOXO6, FSTL1, FUCA2,GAL, GART, GAS5, GDPD5, GLCE, GLI2, GLIS1, GLIS3, GLT25D2, GNA14, GNAS,GPR123, GPX7, GRM5, GTF2H5, GUCY1A3, H2AFJ, HCG 1776018, HDAC4, HEBP2,HELQ, HERPUD2, HHLA3, H1ST1H2AC, HIST1H2BD, HIST1H2BK, HIST2H2BE,HMGCLL1, HNRNPR, HPCAL1, HPDL, HPS3, HSBP1L1, HSPA1A, HTATIP2, ICAM2,IFNAR1, IGFBP5, IGFBP7, IGSF5, IL10RB, IL13RA2, IL17D, IL20RA, IL7,IMMP2L, INSR, IRS1, ITGB1BP1, JMY, JRKL, KAT2B, KBTBD11, KCMF1, KCNMA1,KCNQ2, KCNQ5, KCTD18, KDM1A, KDSR, KHDC1, KIAA1109, KIAA1324, KIAA1598,KIAA1804, KIRREL3, KLHDC1, KRCC1, LGALS3, LGALS3BP, LIFR, LINGO1, LIPT1,LMAN2L, LMCD1, LOC100129195, LOC100129884, LOC100130522, LOC100130856,LOC100132167, LOC100216479, LOC100272217, LOC100287039, LOC151146,LOC153682, LOC220930, LOC254128, LOC255167, LOC283588, LOC285286,LOC285550, LOC285878, LOC286052, LOC339290, LOC401321, LOC645513,LOC80154, LOC90246, LOC93622, LOC94431, LPAR3, LPHN2, LRCH2, LRP2BP,LYRM1, LYRM5, MAGI2, MANSC1, MAP3K13, MAP3K5, MAP9, MAPKAP1, MBD5,MBLAC2, MBNL1, MCTP1, MED6, MEGF11, MEOX2, MET, METT5D1, MFSD4, MFSD6,MFSD9, MGAT4A, MORC3, MREG, MRPS18C, MRPS33, MST1, MTMR3, MTUS2, MXRA7,N4BP3, NAP1L3, NCAM1, NCAM2, NCOA7, NCRNA00081, NCRNA00117, NCRNA00171,NDUFA4L2, NDUFV3, NEAT1, NEBL, NEIL2, NETO1, NFATC4, NHEDC2, NIPAL3,NLRX1, NNAT, NOTCH4, NPW, NPY, NR1H3, NR2F1, NT5E, NUDT19, NUDT6,OGFRL1, OLFM3, OMA1, OPRM1, OPTN, P4HTM, PABPC4L, PABPC5, PAK1, PAM,PAPOLG, PAPPA, PBX1, PCDHB10, PCDHB16, PCNX, PCNXL2, PDCD2, PDE4DIP,PDGFRB, PEX12, PEX6, PHKA1, PHLDB3, PIBF1, PID1, PIGH, PIGP, PIK3CB,PIWIL2, PKNOX1, PKNOX2, PLA2G12A, PLCB1, PLEKHA2, PLEKHA3, PLIN2, PLRG1,POLR3GL, POU6F1, PPAPDC1A, PPOX, PPP1R14A, PPP2R5C, PPP3CA, PRAME,PRKACB, PRMT2, PRPH, PSD3, PSTK, PTGER2, PTGES, PTN, PTP4A3, PTPRD,PTPRN2, PTPRR, QPCT, RAB4A, RAB6B, RAC2, RAI2, RCAN1, RCC1, RDH13,RFPL1S, RG9MTD2, RGAG4, RHBDD1, RHBDF2, RHOU, RNF13, RNF41, RNLS, RPL31,RPL37, RPRD1A, RRN3, RSL1D1, RSPH3, SAP30, SAV1, SCG5, SCN5A, SERTAD4,SGMS1, SH3BGR, SH3GL2, SH3 KBP1, SH3YL1, SIAE, SIGIRR, SIK1, SIK3, SIX2,SLC12A7, SLC16A14, SLC22A17, SLC22A5, SLC25A12, SLC25A4, SLC35F3,SLC6A15, SLIT1, SMARCA2, SMEK2, SNAP91, SNCA, SOCS5, SORCS2, SPATA17,SPATA7, SPIN3, SPINT1, SPINT2, SPOCK2, SPRED1, SSPN, ST8SIA3, STAC2,STAR, STEAP3, STOX2, STX12, STXBP5L, SUCLG2, SYNJ1, SYNPR, SYT13, TAF12,TAF1B, TBC1D12, TBC1D15, TCEAL2, TEX264, THAP2, TM2D1, TMCC1, TMEM182,TMEM184C, TMEM196, TMEM45B, TMEM5, TMEM59L, TMEM65, TMIE, TNC,TNFRSF10D, TNFSF4, TOX, TRAPPC9, TRHDE, TRIM61, TRIM69, TSPAN7, TSPAN9,TTC23, TTC39C, TUSC3, TXLNB, UBE2D3, UBE2W, UCN, UNC5A, UTP23, VPS37A,VSTM2A, WASF3, WDFY3, WDTC1, XPR1, XYLT1, ZBTB41, ZC3H12B, ZNF148,ZNF185, ZNF22, ZNF23, ZNF25, ZNF250, ZNF280D, ZNF285A, ZNF295, ZNF346,ZNF528, ZNF585A, ZNF610, ZNF641, ZNF662, ZNF677, and/or ZNF862, ascompared to the expression levels of these genes in cells from theparental SiMa cell line DSMZ ACC 164.

In an aspect of this embodiment, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit at least a 1.5-folddecrease in gene expression levels of one or more genes selected fromACOT1, ADARB1, ASAM, BACE2, C11ORF70, C2CD4A, C3ORF23, CADM2, CAP2,CARTPT, CDKN1C, CFC1, CNGA3, COL5A1, CYGB, CYP2E1, CYTL1, DSCAM, ECEL1,ECHDC3, FAM26F, FAM49A, FGF1, FGF13, FGF19, FLJ39051, FUCA2, GAL, GPX7,GRM5, HCG 1776018, HHLA3, H1ST1H2AC, HIST1H2BD, HSPA1A, HTATIP2, ICAM2,IGFBP5, IGFBP7, IL13RA2, KCNMA1, KCNQ5, KIAA1598, KRCC1, LMCD1,LOC100216479, LOC254128, LOC339290, LPAR3, MEGF11, MEOX2, NCAM2,NDUFA4L2, NEAT1, NNAT, NPW, NPY, PABPC4L, PAPPA, PID1, PPAPDC1A, PRAME,PRKACB, PTGER2, PTN, PTP4A3, RAC2, SLC35F3, SYT13, TCEAL2, THAP2, TMIE,TNFRSF10D, TRHDE, TXLNB, and/or ZNF662, as compared to the expressionlevels of these genes in cells from the parental SiMa cell line DSMZ ACC164.

In another aspect of this embodiment, cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit at least a 1.5-folddecrease in gene expression levels of one or more genes selected fromACOT1, ASAM, BACE2, C11ORF70, C2CD4A, C3ORF23, CADM2, CAP2, CARTPT,CFC1, CNGA3, CYGB, CYP2E1, CYTL1, DSCAM, ECEL1, ECHDC3, FGF1, FGF13,FUCA2, GAL, GPX7, HSPA1A, HTATIP2, ICAM2, IGFBP5, IGFBP7, KIAA1598,KRCC1, LMCD1, LOC100216479, LOC254128, LOC339290, LPAR3, MEOX2,NDUFA4L2, NEAT1, NNAT, NPW, NPY, PAPPA, PID1, PPAPDC1A, PRAME, PTN,SLC35F3, SYT13, TCEAL2, TMIE, TNFRSF10D, TRHDE, and/or ZNF662, ascompared to the expression levels of these genes in cells from theparental SiMa cell line DSMZ ACC 164.

In yet another aspect of this embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit at least a1.5-fold decrease in gene expression levels of one or more genesselected from C11ORF70, CADM2, CAP2, CARTPT, CNGA3, CYGB, CYP2E1, CYTL1,DSCAM, FGF13, FUCA2, GAL, HSPA1A, HTATIP2, ICAM2, IGFBP5, IGFBP7, LMCD1,LOC100216479, MEOX2, NDUFA4L2, NEAT1, NNAT, NPW, PID1, PRAME, SLC35F3,SYT13, TCEAL2, TMIE, TNFRSF10D, and/or ZNF662, as compared to theexpression levels of these genes in cells from the parental SiMa cellline DSMZ ACC 164.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes listed in Tables 5 or 7as compared to the expression levels of these genes in cells from the2D6 cell line, and at least a 1.5-fold increase in gene expressionlevels of one or more genes listed in Tables 9 or 11 as compared to theexpression levels of these genes in cells from a parental SiMa cellline. In aspects of this embodiment, cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit at least a 1.5-foldincrease in gene expression levels of one or more genes listed in Tables5 or 7 as compared to the expression levels of these genes in cells fromthe 2D6 cell line, and at least a 1.5-fold increase in gene expressionlevels of one or more genes listed in Tables 9 or 11 as compared to theexpression levels of these genes in cells from the parental SiMa cellline DSMZ ACC 164. In other aspects of this embodiment, cells from anestablished clonal cell line susceptible to BoNT/A intoxication exhibitan increase in gene expression levels of one or more genes listed inTables 5 or 7 of, e.g., at least a 1.5-fold, at least a 2.0-fold, atleast a 2.5-fold, at least a 3.0-fold, at least a 3.5-fold, at least a4.0-fold, at least a 4.5-fold, at least a 5.0-fold, at least a 5.5-fold,at least a 6.0-fold, at least 7.0-fold, or at least 8.0-fold as comparedto the expression levels of these genes in cells from the 2D6 cell line,and exhibit an increase in gene expression levels of one or more geneslisted in Tables 9 or 11 of, e.g., at least a 1.5-fold, at least a2.0-fold, at least a 2.5-fold, at least a 3.0-fold, at least a 3.5-fold,at least a 4.0-fold, at least a 4.5-fold, at least a 5.0-fold, at leasta 5.5-fold, at least a 6.0-fold, at least 7.0-fold, or at least 8.0-foldas compared to the expression levels of these genes in cells from theparental SiMa cell line DSMZ ACC 164. In other aspects of thisembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit an increase in gene expression levels of,e.g., 2 or more genes, 3 or more genes, 4 or more genes, 5 or moregenes, 6 or more genes, 7 or more genes, 8 or more genes, 9 or moregenes, 10 or more genes, 20 or more genes, 30 or more genes, 40 or moregenes, 50 or more genes, 60 or more genes, 70 or more genes, 80 or moregenes, 90 or more genes, or 100 or more genes listed in Tables 5 or 7 ascompared to the expression levels of these genes in cells from the 2D6cell line, and listed in Tables 9 or 11 as compared to the expressionlevels of these genes in cells from the parental SiMa cell line DSMZ ACC164. In yet other aspects cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit an increase in geneexpression levels of, e.g., about 5 genes to about 100 genes, about 10genes to about 100 genes, about 15 genes to about 100 genes, about 20genes to about 100 genes, about 25 genes to about 100 genes, about 5genes to about 75 genes, about 10 genes to about 75 genes, about 15genes to about 75 genes, about 20 genes to about 75 genes, about 25genes to about 75 genes, about 5 genes to about 50 genes, about 10 genesto about 50 genes, about 15 genes to about 50 genes, about 20 genes toabout 50 genes, or about 25 genes to about 50 genes listed in Tables 5or 7 as compared to the expression levels of these genes in cells fromthe 2D6 cell line, and listed in Tables 9 or 11 as compared to theexpression levels of these genes in cells from the parental SiMa cellline DSMZ ACC 164.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from A2BP1,ALCAM, ANAPC7, ARF1, ARHGAP6, ARHGEF6, ARL17A, ASCL1, ASXL3, BASP1,BNC2, C10ORF58, C12ORF49, C1ORF43, C20ORF7, CBLB, CBWD1, CCDC3, CD9,CDC25B, CHRNA7, CPVL, CRYBG3, CSE1L, CUGBP2, CXCR4, DACT1, DAZ1, DBH,DHX35, DKFZP434L187, DLEU2, DLL3, EAF2, EFNA5, EIF3C, ELOVL7, EML1,ENC1, EXOSC6, EXPH5, EZH2, FAM181B, FAM19A4, FAM7A3, FUBP3, FZD5, GAP43,GFRA2, GNAI1, GNB4, GNG11, GPR125, GRP, GSS, ITGA6, ITGA6, ITGB5,KCTD12, KDELC2, KHDRBS3, KIF16B, KLHL13, LBH, LOC100128844, LOC340109,LOC641298, LOC728052, LUM, MAB21L1, MAN2A1, MAOA, MINA, MMD, MSN, MYO1B,MYO6, NAAA, NEDD9, NOS1, OSBPL3, PCGF5, PELI2, PFKFB3, PHF20, PLK2,PLS3, PLXNA2, PLXNA4, PM20D2, POLE, PSMA7, PTGFRN, PTPRE, PTPRG, RAB35,RAF1, RAN, RASEF, RBL1, RELL1, RGS5, RIMBP2, RNF182, RNF34, S1PR3,SALL4, SEMA6A, SEZ6L, SIM1, SLC44A5, SLC7A2, SLCO3A1, SLITRK5, SPAG6,SPATS2L, ST8SIA1, STMN3, STRA6, TCF7L2, TFPI2, TH1L, THOC4, TIMP3, TLE3,TMEM132C, TMEM178, TPBG, TRA2A, TRIM29, TRIM36, TSHZ3, TXNRD1, U2AF1,UBE2V1, VPS29, ZFHX4, ZGPAT, ZNF217, and/or ZNF503, as compared to theexpression levels of these genes in cells from both the 2D6 cell lineand the parental SiMa cell line DSMZ ACC 164.

In an aspect of this embodiment, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit at least a 1.5-foldincrease in gene expression levels of one or more genes selected fromALCAM, ARHGAP6, ARHGEF6, ASCL1, BNC2, CCDC3, CHRNA7, CRYBG3, CUGBP2,CXCR4, DAZ1, EFNA5, ELOVL7, EML1, ENC1, EXPH5, FAM7A3, FZD5, GFRA2,GNAI1, GNB4, GNG11, GRP, ITGA6, KCTD12, LOC100128844, LOC340109,LOC728052, LUM, MAB21L1, MAB21L2, MAOA, MSN, NEDD9, OSBPL3, PLK2,PTGFRN, PTPRE, RGS5, RNF182, SIM1, SLC44A5, SLITRK5, SPAG6, TFPI2,TIMP3, TMEM132C, TPBG, TRIM29, and/or TRIM36, as compared to theexpression levels of these genes in cells from both the 2D6 cell lineand the parental SiMa cell line DSMZ ACC 164.

In another aspect of this embodiment, cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit at least a 1.5-foldincrease in gene expression levels of one or more genes selected fromALCAM, ARHGAP6, ARHGEF6, CCDC3, CHRNA7, CRYBG3, CUGBP2, CXCR4, DAZ1,ELOVL7, EML1, EXPH5, FAM7A3, GNB4, GNG11, GRP, ITGA6, KCTD12, LOC340109,LUM, MAB21L1, MAB21L2, PLK2, PTPRE, RGS5, RNF182, SIM1, SLC44A5,SLITRK5, SPAG6, TIMP3, and/or TMEM132C, as compared to the expressionlevels of these genes in cells from both the 2D6 cell line and theparental SiMa cell line DSMZ ACC 164.

In yet another aspect of this embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit at least a1.5-fold increase in gene expression levels of one or more genesselected from CCDC3, CHRNA7, CRYBG3, CXCR4, DAZ1, EXPH5, FAM7A3, GNB4,GRP, KCTD12, LUM, PLK2, PTPRE, RGS5, SIM1, SLITRK5, TIMP3, and/orTMEM132C, as compared to the expression levels of these genes in cellsfrom both the 2D6 cell line and the parental SiMa cell line DSMZ ACC164.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from A2BP1,ALCAM, ARF1, ARL17A, ASCL1, BASP1, CD9, CUGBP2, EAF2, EIF3C, FAM181B,FUBP3, GNAI1, GNB4, GNG11, KDELC2, KHDRBS3, KLHL13, LOC641298,LOC728052, MAB21L2, MAOA, MINA, MSN, MYO6, PLK2, PLS3, RAF1, RELL1,RNF182, SEZ6L, SEZ6L, SIM1, SLC44A5, SLC7A2, SLCO3A1, SLITRK5, SPATS2L,TCF7L2, TFPI2, TMEM178, TRA2A, U2AF1, and/or ZNF217, as compared to theexpression levels of these genes in cells from both the 2D6 cell lineand the parental SiMa cell line DSMZ ACC 164.

In an aspect of this embodiment, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit at least a 1.5-foldincrease in gene expression levels of one or more genes selected fromA2BP1, ALCAM, ARF1, ASCL1, BASP1, CD9, CUGBP2, EIF3C, FUBP3, GNAI1,GNB4, GNG11, KDELC2, KHDRBS3, LOC641298, LOC728052, MAB21L2, MAOA, MINA,MSN, MYO6, PLK2, PLS3, RELL1, RNF182, SIM1, SLC44A5, SLC7A2, SLCO3A1,SLITRK5, TCF7L2, TFPI2, TMEM178, TRA2A, and/or ZNF217, as compared tothe expression levels of these genes in cells from both the 2D6 cellline and the parental SiMa cell line DSMZ ACC 164.

In another aspect of this embodiment, cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit at least a 1.5-foldincrease in gene expression levels of one or more genes selected fromASCL1, CUGBP2, GNAI1, GNB4, GNG11, LOC728052, MAB21L2, MAOA, MSN, PLK2,RNF182, SIM1, SLC44A5, SLITRK5, and/or TFPI2, as compared to theexpression levels of these genes in cells from both the 2D6 cell lineand the parental SiMa cell line DSMZ ACC 164.

In yet another aspect of this embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit at least a1.5-fold increase in gene expression levels of one or more genesselected from GNB4, GNG11, PLK2, RNF182, SIM1, SLC44A5, and/or SLITRK5,as compared to the expression levels of these genes in cells from boththe 2D6 cell line and the parental SiMa cell line DSMZ ACC 164.

In still another aspect of this embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit at least a1.5-fold increase in gene expression levels of one or more genesselected from GNB4, PLK2, SIM1, and/or SLITRK5, as compared to theexpression levels of these genes in cells from both the 2D6 cell lineand the parental SiMa cell line DSMZ ACC 164.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold decreasein gene expression levels of one or more genes listed in Tables 6 or 8as compared to the expression levels of these genes in cells from the2D6 cell line, and at least a 1.5-fold decrease in gene expressionlevels of one or more genes listed in Tables 10 or 12 as compared to theexpression levels of these genes in cells from a parental SiMa cellline. In aspects of this embodiment, cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit at least a 1.5-folddecrease in gene expression levels of one or more genes listed in Tables6 or 8 as compared to the expression levels of these genes in cells fromthe 2D6 cell line, and at least a 1.5-fold decrease in gene expressionlevels of one or more genes listed in Tables 10 or 12 as compared to theexpression levels of these genes in cells from the parental SiMa cellline DSMZ ACC 164. In other aspects of this embodiment, cells from anestablished clonal cell line susceptible to BoNT/A intoxication exhibita decrease in gene expression levels of one or more genes listed inTables 6 or 8 of, e.g., at least a 1.5-fold, at least a 2.0-fold, atleast a 2.5-fold, at least a 3.0-fold, at least a 3.5-fold, at least a4.0-fold, at least a 4.5-fold, at least a 5.0-fold, at least a 5.5-fold,at least a 6.0-fold, at least 7.0-fold, or at least 8.0-fold as comparedto the expression levels of these genes in cells from the 2D6 cell line,and a decrease in gene expression levels of one or more genes listed inTables 10 or 12 of, e.g., at least a 1.5-fold, at least a 2.0-fold, atleast a 2.5-fold, at least a 3.0-fold, at least a 3.5-fold, at least a4.0-fold, at least a 4.5-fold, at least a 5.0-fold, at least a 5.5-fold,at least a 6.0-fold, at least 7.0-fold, or at least 8.0-fold as comparedto the expression levels of these genes in cells from the parental SiMacell line DSMZ ACC 164. In other aspects of this embodiment, cells froman established clonal cell line susceptible to BoNT/A intoxicationexhibit a decrease in gene expression levels of, e.g., 2 or more genes,3 or more genes, 4 or more genes, 5 or more genes, 6 or more genes, 7 ormore genes, 8 or more genes, 9 or more genes, 10 or more genes, 20 ormore genes, 30 or more genes, 40 or more genes, 50 or more genes, 60 ormore genes, 70 or more genes, 80 or more genes, 90 or more genes, or 100or more genes listed in Tables 6 or 8 as compared to the expressionlevels of these genes in cells from the 2D6 cell line, and listed inTables 10 or 12 as compared to the expression levels of these genes incells from the parental SiMa cell line DSMZ ACC 164. In yet otheraspects cells from an established clonal cell line susceptible to BoNT/Aintoxication exhibit a decrease in gene expression levels of, e.g.,about 5 genes to about 100 genes, about 10 genes to about 100 genes,about 15 genes to about 100 genes, about 20 genes to about 100 genes,about 25 genes to about 100 genes, about 5 genes to about 75 genes,about 10 genes to about 75 genes, about 15 genes to about 75 genes,about 20 genes to about 75 genes, about 25 genes to about 75 genes,about 5 genes to about 50 genes, about 10 genes to about 50 genes, about15 genes to about 50 genes, about 20 genes to about 50 genes, or about25 genes to about 50 genes listed in Tables 6 or 8 as compared to theexpression levels of these genes in cells from the 2D6 cell line, andlisted in Tables 10 or 12 as compared to the expression levels of thesegenes in cells from the parental SiMa cell line DSMZ ACC 164.

In an embodiment, cells from an established clonal cell line susceptibleto BoNT/A intoxication exhibit at least a 1.5-fold decrease in geneexpression levels of one or more genes selected from ABTB1, ACOT1,ACYP2, ADARB1, ADCY1, ANKRD50, ASAM, ASPHD1, ATP2B3, BACE1, BRUNOL4,BRWD1, BTN3A3, C16ORF52, C1ORF21, C21ORF57, C2CD2, C2CD4A, C3ORF23,C9ORF150, CAMK2N2, CAP2, CAPN2, CARTPT, CD302, CDH12, CDKN2A, CDS1,CFC1, CLCN3, CLDN12, CLMN, CNGA3, CNNM1, CNOT6L, COL6A1, CYGB, CYLD,CYP2E1, CYTL1, D4S234E, DCLK1, DCTD, DUSP16, EFNB3, ERAP1, ERBB2, ERBB4,ETFDH, FAM162B, FAM165B, FAM46A, FBXW7, FGD5, FGF13, FOXO6, FSTL1, GAL,GLT25D2, GNAS, GPR123, GPX7, GRM5, GUCY1A3, HCG 1776018, HELQ, HERPUD2,H1ST1H2AC, HIST1H2BD, HIST1H2BK, HIST2H2BE, HNRNPR, HPCAL1, IL10RB, JMY,KAT2B, KBTBD11, KCNMA1, KCNQ2, KHDC1, KIAA1598, KLHDC1, LGALS3BP, LIFR,LOC100130522, LOC94431, LRP2BP, LYRM5, MAP3K5, MAP9, MCTP1, MEOX2,MFSD4, MGAT4A, MRPS33, MST1, NAP1L3, NCOA7, NCRNA00171, NDUFA4L2,NDUFV3, NEBL, NHEDC2, NIPAL3, NLRX1, NUDT19, OLFM3, PAM, PAPPA, PCNXL2,PDGFRB, PHKA1, PIGH, PLRG1, PLRG1, PPAPDC1A, PRKACB, PRPH, PTGER2, PTN,PTPRD, RAB6B, RGAG4, RHOU, RNF13, RNF41, RSL1D1, SH3GL2, SH3YL1, SIGIRR,SIK1, SLC12A7, SLC22A17, SLC35F3, SNCA, SPINT2, SPOCK2, ST8SIA3, STAR,STEAP3, STOX2, SYNJ1, SYT13, TM2D1, TMEM184C, TMEM5, TMEM59L, TMIE,TTC39C, TUSC3, TXLNB, UCN, WDFY3, WDFY3, ZNF641, and/or ZNF662, ascompared to the expression levels of these genes in cells from both the2D6 cell line and the parental SiMa cell line DSMZ ACC 164.

In an aspect of this embodiment, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit at least a 1.5-folddecrease in gene expression levels of one or more genes selected fromACOT1, ADARB1, ASAM, C2CD4A, C3ORF23, CAP2, CARTPT, CFC1, CNGA3, CYGB,CYP2E1, CYTL1, FGF13, GAL, GPX7, GRM5, HCG 1776018, H1ST1H2AC,HIST1H2BD, KCNMA1, KIAA1598, MEOX2, NDUFA4L2, PPAPDC1A, PRKACB, PTGER2,PTN, SLC35F3, SYT13, TMIE, TXLNB, and/or ZNF662, as compared to theexpression levels of these genes in cells from both the 2D6 cell lineand the parental SiMa cell line DSMZ ACC 164.

In another aspect of this embodiment, cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit at least a 1.5-folddecrease in gene expression levels of one or more genes selected fromACOT1, ASAM, C2CD4A, C3ORF23, CAP2, CARTPT, CFC1, CNGA3, CYGB, CYP2E1,CYTL1, FGF13, GAL, GPX7, KIAA1598, MEOX2, NDUFA4L2, PPAPDC1A, PTN,SLC35F3, SYT13, TMIE, and/or ZNF662, as compared to the expressionlevels of these genes in cells from both the 2D6 cell line and theparental SiMa cell line DSMZ ACC 164.

In yet another aspect of this embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit at least a1.5-fold decrease in gene expression levels of one or more genesselected from CAP2, CARTPT, CNGA3, CYGB, CYP2E1, CYTL1, FGF13, GAL,MEOX2, NDUFA4L2, SLC35F3, SYT13, TMIE, and/or ZNF662, as compared to theexpression levels of these genes in cells from both the 2D6 cell lineand the parental SiMa cell line DSMZ ACC 164.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold decreasein gene expression levels of one or more genes selected from CAP2,CDKN2A, CNGA3, FAM162B, FGF13, FOXO6, FSTL1, KCNMA1, KHDC1, LIFR, MCTP1,MEOX2, PPAPDC1A, PTN, SYT13, TMIE, and/or ZNF662, as compared to theexpression levels of these genes in cells from both the 2D6 cell lineand the parental SiMa cell line DSMZ ACC 164.

In an aspect of this embodiment, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit at least a 1.5-folddecrease in gene expression levels of one or more genes selected fromCAP2, CDKN2A, CNGA3, FGF13, FOXO6, FSTL1, KCNMA1, KHDC1, MCTP1, MEOX2,PPAPDC1A, PTN, SYT13, TMIE, and/or ZNF662, as compared to the expressionlevels of these genes in cells from both the 2D6 cell line and theparental SiMa cell line DSMZ ACC 164.

In another aspect of this embodiment, cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit at least a 1.5-folddecrease in gene expression levels of one or more genes selected fromCNGA3, FGF13, KCNMA1, MEOX2, PPAPDC1A, PTN, SYT13, TMIE, and/or ZNF662,as compared to the expression levels of these genes in cells from boththe 2D6 cell line and the parental SiMa cell line DSMZ ACC 164.

In yet another aspect of this embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit at least a1.5-fold decrease in gene expression levels of one or more genesselected from CNGA3, FGF13, MEOX2, PPAPDC1A, PTN, SYT13, TMIE, and/orZNF662, as compared to the expression levels of these genes in cellsfrom both the 2D6 cell line and the parental SiMa cell line DSMZ ACC164.

In still another aspect of this embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit at least a1.5-fold decrease in gene expression levels of one or more genesselected from CNGA3, FGF13, MEOX2, SYT13, TMIE, and/or ZNF662, ascompared to the expression levels of these genes in cells from both the2D6 cell line and the parental SiMa cell line DSMZ ACC 164.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-folddifference in gene expression levels of one or more genes listed inTables 14 or 16 as compared to the expression levels of these genes incells from both the 2D6 cell line and the parental SiMa cell line DSMZACC 164. In aspects of this embodiment, cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit at least a 1.5-folddifference in gene expression levels of one or more genes listed inTables 14 or 16 as compared to the expression levels of these genes incells from both the 2D6 cell line and the parental SiMa cell line DSMZACC 164. In other aspects of this embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit a differencein gene expression levels of one or more genes listed in Tables 14 or 16of, e.g., at least a 1.5-fold, at least a 2.0-fold, at least a 2.5-fold,at least a 3.0-fold, at least a 3.5-fold, at least a 4.0-fold, at leasta 4.5-fold, at least a 5.0-fold, at least a 5.5-fold, at least a6.0-fold, at least a 7.0-fold, or at least a 8.0-fold as compared to theexpression levels of these genes in cells from both the 2D6 cell lineand the parental SiMa cell line DSMZ ACC 164. In other aspects of thisembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit an increase in gene expression levels of,e.g., 2 or more genes, 3 or more genes, 4 or more genes, 5 or moregenes, 6 or more genes, 7 or more genes, 8 or more genes, 9 or moregenes, 10 or more genes, 20 or more genes, 30 or more genes, 40 or moregenes, 50 or more genes, 60 or more genes, 70 or more genes, 80 or moregenes, 90 or more genes, or 100 or more genes listed in Tables 14 or 16as compared to the expression levels of these genes in cells from boththe 2D6 cell line and the parental SiMa cell line DSMZ ACC 164. In yetother aspects cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit an increase in gene expression levels of,e.g., about 5 genes to about 100 genes, about 10 genes to about 100genes, about 15 genes to about 100 genes, about 20 genes to about 100genes, about 25 genes to about 100 genes, about 5 genes to about 75genes, about 10 genes to about 75 genes, about 15 genes to about 75genes, about 20 genes to about 75 genes, about 25 genes to about 75genes, about 5 genes to about 50 genes, about 10 genes to about 50genes, about 15 genes to about 50 genes, about 20 genes to about 50genes, or about 25 genes to about 50 genes listed in Tables 14 or 16 ascompared to the expression levels of these genes in cells from both the2D6 cell line and the parental SiMa cell line DSMZ ACC 164.

In an embodiment, cells from an established clonal cell line susceptibleto BoNT/A intoxication exhibit at least a 1.5-fold difference in geneexpression levels of one or more genes selected from ACOT9, ADAMTS9,ARHGAP24, ARHGEF3, ASCL1, BARD1, BASP1, C3ORF70, C11ORF75, CCDC109B,CD9, CDCA7L, CDK2, CENPL, CLSTN2, CNTN1, CSRP2, CTSL2, CUGBP2, DEPDC1,DIAPH3, DOK5, DPYD, DYNLT3, EMILIN2, ETV1, FAM101B, FBLN1, FGFR2, FNDC5,GNAI1, GNB4, GNG11, GNG12, GPR177, GTSE1, HGF, KITLG, LPAR1, MAB21L2,MAOA, MCM10, MINA, MSN, MYO6, MYRIP, PAG1, PEG3, PLK2, POLA2, PPP1R3C,PRLHR, PRSS12, PTGR1, PTPRK, PVRL3, RAB32, RBPMS, SDC2, SGOL2, SLC43A3,SLC7A2, SMC2, SMC6, SPARC, SPC25, ST8SIA4, TCF7L1, TFPI2, TMEM35,TMEM178, TNFAIP8, TPTE, TRIP10, TWIST1, and/or ZNF521, as compared tothe expression levels of these genes in cells from both the 2D6 cellline and the parental SiMa cell line DSMZ ACC 164. In another aspect ofthis embodiment, cells from an established clonal cell line susceptibleto BoNT/A intoxication exhibit at least a 1.5-fold difference in geneexpression levels of one or more genes selected from ADAMTS9, ARHGAP24,ASCL1, BARD1, BASP1, BVES, C11ORF75, CDCA7L, CNTN1, CUGBP2, DOK5, DPYD,DYNLT3, FBLN1, FGFR2, GNAI1, GNB4, GNG11, GNG12, GTSE1, GTSF1, ITPRIP,KDELC2, LOC728052, LPAR1, MAB21L2, MAOA, MINA, MSN, PEG3, PLK2, PRLHR,PRSS12, PTPRK, RBPMS, RNF182, SGOL2, SLC43A3, SLC44A5, SLC7A2, SMC6,SPARC, TFPI2, TMEM178, and/or TPTE, as compared to the expression levelsof these genes in cells from both the 2D6 cell line and the parentalSiMa cell line DSMZ ACC 164. In yet another aspect of this embodiment,cells from an established clonal cell line susceptible to BoNT/Aintoxication exhibit at least a 1.5-fold difference in gene expressionlevels of one or more genes selected from ADAMTS9, ASCL1, BASP1, DOK5,DPYD, GNB4, GNG11, GTSF1, MAOA, MINA, MSN, PEG3, PLK2, PRSS12, RNF182,SLC44A5, SPARC, TFPI2, and/or TPTE, as compared to the expression levelsof these genes in cells from both the 2D6 cell line and the parentalSiMa cell line DSMZ ACC 164.

In another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from ADAMTS9,ARHGAP24, ARHGEF3, ASCL1, BARD1, CD9, CDK2, CSRP2, CTSL2, DIAPH3, DOK5,DYNLT3, EMILIN2, ETV1, FBLN1, FGFR2, GNAI1, GNB4, GNG11, GNG12, HGF,KITLG, LPAR1, MCM10, MSN, PAG1, PEG3, PLK2, POLA2, PPP1R3C, PTPRK,RAB32, SDC2, SLC43A3, SLC7A2, SMC6, SPARC, SPC25, ST8SIA4, TCF7L1,TFPI2, TMEM35, TNFAIP8, TPTE, TRIP10, and/or TWIST1, as compared to theexpression levels of these genes in cells from both the 2D6 cell lineand the parental SiMa cell line DSMZ ACC 164. In an aspect of thisembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit at least a 1.5-fold increase in geneexpression levels of one or more genes selected from ADAMTS9, ARHGAP24,ASCL1, BARD1, DOK5, DYNLT3, FBLN1, FGFR2, GNAI1, GNB4, GNG11, GNG12,LPAR1, MSN, PEG3, PLK2, PTPRK, SLC43A3, SLC7A2, SMC6, SPARC, TFPI2,and/or TPTE, as compared to the expression levels of these genes incells from both the 2D6 cell line and the parental SiMa cell line DSMZACC 164. In another aspect of this embodiment, cells from an establishedclonal cell line susceptible to BoNT/A intoxication exhibit at least a1.5-fold increase in gene expression levels of one or more genesselected from ADAMTS9, ASCL1, DOK5, GNB4, GNG11, MSN, PEG3, PLK2, SPARC,TFPI2, and/or TPTE, as compared to the expression levels of these genesin cells from both the 2D6 cell line and the parental SiMa cell lineDSMZ ACC 164.

In yet another embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit at least a 1.5-fold increasein gene expression levels of one or more genes selected from ACOT9,BASP1, C11ORF75, CCDC109B, CDCA7L, CLSTN2, CNTN1, CUGBP2, DEPDC1, DPYD,FAM101B, FNDC5, GTSE1, MAOA, MINA, MYO6, MYRIP, PLK2, PRLHR, PVRL3,RBPMS, SGOL2, SMC2, TFP12, TMEM178, and/or ZNF521, as compared to theexpression levels of these genes in cells from both the 2D6 cell lineand the parental SiMa cell line DSMZ ACC 164. In an aspect of thisembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit at least a 1.5-fold increase in geneexpression levels of one or more genes selected from BASP1, C11ORF75,CDCA7L, CNTN1, CUGBP2, DPYD, GTSE1, MAOA, MINA, PLK2, PRLHR, RBPMS,SGOL2, TFPI2, and/or TMEM178, as compared to the expression levels ofthese genes in cells from both the 2D6 cell line and the parental SiMacell line DSMZ ACC 164. In another aspect of this embodiment, cells froman established clonal cell line susceptible to BoNT/A intoxicationexhibit at least a 1.5-fold increase in gene expression levels of one ormore genes selected from BASP1, DPYD, MAOA, MINA, PLK2, and/or TFPI2, ascompared to the expression levels of these genes in cells from both the2D6 cell line and the parental SiMa cell line DSMZ ACC 164.

In a still another embodiment, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit at least a 1.5-foldincrease in gene expression levels of one or more genes selected fromC3ORF70, MAB21L2, PRSS12, CENPL, GPR177, and/or PTGR1, as compared tothe expression levels of these genes in cells from both the 2D6 cellline and the parental SiMa cell line DSMZ ACC 164. In an aspect of thisembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit at least a 1.5-fold increase in geneexpression levels of one or more genes selected from MAB21L2 and/orPRSS12, as compared to the expression levels of these genes in cellsfrom both the 2D6 cell line and the parental SiMa cell line DSMZ ACC164.

Aspects of the present disclosure comprise, in part, cells from anestablished clonal cell line susceptible to BoNT/A intoxication thatexhibit an equivalent or lower EC₅₀ for BoNT/A activity relative to theEC₅₀ for BoNT/A activity of cells from a parental SiMa cell line. In anaspect of this embodiment, cells from an established clonal cell linesusceptible to BoNT/A intoxication exhibit an equivalent or lower EC₅₀for BoNT/A activity relative to the EC₅₀ for BoNT/A activity of cellsfrom the parental SiMa cell line DSMZ ACC 164. In an aspect of thisembodiment, cells from an established clonal cell line susceptible toBoNT/A intoxication exhibit an equivalent EC₅₀ for BoNT/A activityrelative to the EC₅₀ for BoNT/A activity of cells from the parental SiMacell line DSMZ ACC 164. In aspects of this embodiment, cells from anestablished clonal cell line susceptible to BoNT/A intoxication exhibita lower EC₅₀ for BoNT/A activity of, e.g., at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 100% relative to the EC₅₀ forBoNT/A activity of cells from the parental SiMa cell line DSMZ ACC 164.In other aspects of this embodiment, cells from an established clonalcell line susceptible to BoNT/A intoxication exhibit a lower EC₅₀ forBoNT/A activity of, e.g., at least 110%, at least 120%, at least 130%,at least 140%, at least 150%, at least 160%, at least 170%, at least180%, at least 190%, or at least 200% relative to the EC₅₀ for BoNT/Aactivity of cells from the parental SiMa cell line DSMZ ACC 164. In yetother aspects of this embodiment, cells from an established clonal cellline susceptible to BoNT/A intoxication exhibit a lower EC₅₀ for BoNT/Aactivity of, e.g., at least 225%, at least 250%, at least 275%, at least300%, at least 325%, at least 350%, at least 375%, at least 400%, atleast 425%, at least 450%, at least 475%, or at least 500% relative tothe EC₅₀ for BoNT/A activity of cells from the parental SiMa cell lineDSMZ ACC 164. In still other aspects of this embodiment, cells from anestablished clonal cell line susceptible to BoNT/A intoxication exhibitan EC₅₀ for BoNT/A activity of, e.g., 10 pM or less, 9 pM or less, 8 pMor less, 7 pM or less, 6 pM or less, 5 pM or less, 4 pM or less, 3.0 pMor less, 2.9 pM or less, 2.8 pM or less, 2.7 pM or less, 2.6 pM or less,2.5 pM or less, 2.4 pM or less, 2.3 pM or less, 2.2 pM or less, 2.1 pMor less, 2.0 pM or less, 1.9 pM or less, 1.8 pM or less, 1.7 pM or less,1.6 pM or less, 1.5 pM or less, 1.4 pM or less, 1.3 pM or less, 1.2 pMor less, 1.1 pM or less, 1.0 pM or less, 0.9 pM or less, 0.8 pM or less,0.7 pM or less, 0.6 pM or less, 0.5 pM or less, 0.4 pM or less, 0.3 pMor less, 0.2 pM or less, or 0.1 pM or less.

Aspects of the present disclosure comprise, in part, a BoNT/A. As usedherein, the term “BoNT/A” is synonymous with “botulinum neurotoxinserotype A” or “botulinum neurotoxin type A” and refers to both anaturally-occurring BoNT/A or a non-naturally occurring BoNT/As thereof,and includes BoNT/A complex comprising the about 150 kDa BoNT/Aneurotoxin and associated non-toxin associated proteins (NAPs), as wellas the about 150 kDa BoNT/A neurotoxin alone. Non-limiting examples ofBoNT/A complexes include, e.g., the 900-kDa BoNT/A complex, the 500-kDaBoNT/A complex, the 300-kDa BoNT/A complex. Non-limiting examples of theabout 150 kDa BoNT/A neurotoxin include, e.g., SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4.

As used herein, the term “naturally occurring BoNT/A” refers to anyBoNT/A produced by a naturally-occurring process, including, withoutlimitation, BoNT/A isoforms produced from a post-translationalmodification, an alternatively-spliced transcript, or a spontaneousmutation, and BoNT/A subtypes, such as, e.g., a BoNT/A1 subtype, BoNT/A2subtype, BoNT/A3 subtype, BoNT/A4 subtype, and BoNT/A5 subtype. Anaturally occurring BoNT/A includes, without limitation, SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or one that substitutes,deletes or adds, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 ormore, 30 or more, 40 or more, 50 or more, or 100 amino acids from SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Commerciallyavailable pharmaceutical compositions of a naturally-occurring BoNT/Aincludes, without limitation, BOTOX® (Allergan, Inc., Irvine, Calif.),DYSPORT®/RELOXIN®, (Ipsen Ltd., Slough, England), PURTOX® (Mentor Corp.,Santa Barbara, Calif.), XEOMIN® (Merz Pharmaceuticals, GmbH., Frankfurt,Germany), NEURONOX® (Medy-Tox, Inc., Ochang-myeon, South Korea), BTX-A.

As used herein, the term “non-naturally occurring BoNT/A” refers to anyBoNT/A whose structure was modified with the aid of human manipulation,including, without limitation, a BoNT/A with an altered amino acidsequence produced by genetic engineering using random mutagenesis orrational design and a BoNT/A produced by in vitro chemical synthesis.Non-limiting examples of non-naturally occurring BoNT/As are describedin, e.g., Steward, L. E. et al., Post-translational Modifications andClostridial Neurotoxins, U.S. Pat. No. 7,223,577; Dolly, J. O. et al.,Activatable Clostridial Toxins, U.S. Pat. No. 7,419,676; Steward, L. E.et al., Clostridial Neurotoxin Compositions and Modified ClostridialNeurotoxins, US 2004/0220386; Steward, L. E. et al., ModifiedClostridial Toxins With Enhanced Targeting Capabilities For EndogenousClostridial Toxin Receptor Systems, U.S. Patent Publication No.2008/0096248; Steward, L. E. et al., Modified Clostridial Toxins WithAltered Targeting Capabilities For Clostridial Toxin Target Cells, U.S.Patent Publication No. 2008/0161543; Steward, L. E. et al., ModifiedClostridial Toxins With Enhanced Translocation Capabilities and AlteredTargeting Activity For Clostridial Toxin Target Cells, U.S. PatentPublication No. 2008/0241881, each of which is hereby incorporated byreference in its entirety.

Thus in an embodiment, the BoNT/A activity being detected is from anaturally occurring BoNT/A. In aspects of this embodiment, the BoNT/Aactivity being detected is from a BoNT/A isoform or a BoNT/A subtype. Inaspects of this embodiment, the BoNT/A activity being detected is fromthe BoNT/A of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.In other aspects of this embodiment, the BoNT/A activity being detectedis from a BoNT/A having, e.g., at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, or at least 95% amino acid identity with SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In other aspectsof this embodiment, the BoNT/A activity being detected is from BOTOX®,DYSPORT®/RELOXIN®, PURTOX®, XEOMIN®, NEURONOX®, or BTX-A.

In another embodiment, the BoNT/A activity being detected is from anon-naturally occurring BoNT/A. In other aspects of this embodiment, theBoNT/A activity being detected is from a non-naturally occurring BoNT/Avariant having, e.g., at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% amino acid identity with SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In other aspects of thisembodiment, the BoNT/A activity being detected is from a non-naturallyoccurring BoNT/A variant having, e.g., 1 or more, 2 or more, 3 or more,4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 ormore, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or morenon-contiguous amino acid substitutions, deletions, or additionsrelative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.In yet other aspects of this embodiment, the BoNT/A activity beingdetected is from a non-naturally occurring BoNT/A variant having, e.g.,1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 ormore, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 ormore, 50 or more, or 100 or more contiguous amino acid substitutions,deletions, or additions relative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, or SEQ ID NO: 4.

Aspects of the present disclosure comprise, in part, a SNAP-25. As usedherein, the term “SNAP-25” refers to a naturally-occurring SNAP-25 or anon-naturally occurring SNAP-25 which is preferentially cleaved by aBoNT/A. As used herein, the term “preferentially cleaved” refers to thatthe cleavage rate of a BoNT/A substrate by a BoNT/A that is at least oneorder of magnitude higher than the cleavage rate of any other substrateby BoNT/A. In aspects of this embodiment, the cleavage rate of a BoNT/Asubstrate by a BoNT/A that is at least two orders of magnitude higher,at least three orders of magnitude higher, at least four orders ofmagnitude higher, or at least five orders of magnitude higher than thecleavage rate of any other substrate by BoNT/A.

As used herein, the term “naturally occurring SNAP-25” refers to anySNAP-25 produced by a naturally-occurring process, including, withoutlimitation, SNAP-25 isoforms produced from a post-translationalmodification, an alternatively-spliced transcript, or a spontaneousmutation, and SNAP-25 subtypes. A naturally occurring SNAP-25 includes,without limitation, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, or SEQ ID NO: 24, or one that substitutes, deletes or adds,e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more,7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40or more, 50 or more, or 100 or more amino acids from SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

As used herein, the term “non-naturally occurring SNAP-25” refers to anySNAP-25 whose structure was modified with the aid of human manipulation,including, without limitation, a SNAP-25 produced by genetic engineeringusing random mutagenesis or rational design and a SNAP-25 produced by invitro chemical synthesis. Non-limiting examples of non-naturallyoccurring SNAP-25s are described in, e.g., Steward, L. E. et al., FRETProtease Assays for Clostridial Toxins, U.S. Pat. No. 7,332,567;Fernandez-Salas et al., Lipohilic Dye-based FRET Assays for ClostridialToxin Activity, U.S. Patent Publication 2008/0160561, each of which ishereby incorporated by reference in its entirety. A non-naturallyoccurring SNAP-25 may substitute, delete or add, e.g., 1 or more, 2 ormore, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more,9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more,or 100 or more amino acids from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

Thus in an embodiment, a SNAP-25 is a naturally occurring SNAP-25. Inaspects of this embodiment, the SNAP-25 is a SNAP-25 isoform or aSNAP-25 subtype. In aspects of this embodiment, the naturally occurringSNAP-25 is the naturally occurring SNAP-25 of SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In otheraspects of this embodiment, the SNAP-25 is a naturally occurring SNAP-25having, e.g., at least 70% amino acid identity, at least 75%, at least80%, at least 85%, at least 90%, or at least 95% amino acid identitywith SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ IDNO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23,or SEQ ID NO: 24.

In another embodiment, a SNAP-25 is a non-naturally occurring SNAP-25.In other aspects of this embodiment, the SNAP-25 is a non-naturallyoccurring SNAP-25 having, e.g., at least 70%, at least 75%, at least80%, at least 85%, at least 90%, or at least 95% amino acid identitywith SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ IDNO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23,or SEQ ID NO: 24. In other aspects of this embodiment, the SNAP-25 is anon-naturally occurring SNAP-25 having, e.g., 1 or more, 2 or more, 3 ormore, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more,10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 ormore non-contiguous amino acid substitutions, deletions, or additionsrelative to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 23, or SEQ ID NO: 24. In yet other aspects of this embodiment, theSNAP-25 is a non-naturally occurring SNAP-25 having, e.g., 1 or more, 2or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 ormore, or 100 or more contiguous amino acid substitutions, deletions, oradditions relative to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,SEQ ID NO: 23, or SEQ ID NO: 24.

A SNAP-25 can be an endogenous SNAP-25 or an exogenous SNAP-25. As usedherein, the term “endogenous SNAP-25” refers to a SNAP-25 naturallypresent in the cell because it is naturally encoded within the cell'sgenome, such that the cell inherently expresses the SNAP-25 without theneed of an external source of SNAP-25 or an external source of geneticmaterial encoding a SNAP-25. The expression of an endogenous SNAP-25 maybe with or without environmental stimulation such as, e.g., celldifferentiation. By definition, an endogenous SNAP-25 can only be anaturally-occurring SNAP-25 or variants thereof. For example, thefollowing established cell lines express an endogenous SNAP-25:BE(2)-M17, Kelly, LA1-55n, N1E-115, N4TG3, N18, Neuro-2a, NG108-15,PC12, SH-SY5Y, SiMa, and SK-N-BE(2)-C.

As used herein, the term “exogenous SNAP-25” refers to a SNAP-25expressed in a cell through the introduction of an external source ofSNAP-25 or an external source of genetic material encoding a SNAP-25 byhuman manipulation. The expression of an exogenous SNAP-25 may be withor without environmental stimulation such as, e.g., celldifferentiation. As a non-limiting example, cells from an establishedclonal cell line can express an exogenous SNAP-25 by transient or stablytransfection of a SNAP-25 coding sequence. As another non-limitingexample, cells from an established clonal cell line can express anexogenous SNAP-25 by protein transfection of a SNAP-25. An exogenousSNAP-25 can be a naturally-occurring SNAP-25 or variants thereof, or anon-naturally occurring SNAP-25 or variants thereof.

Thus in an embodiment, cells from an established clonal cell lineexpress an endogenous SNAP-25. In aspects of this embodiment, theendogenous SNAP-25 expressed by cells from an established clonal cellline is a naturally-occurring SNAP-25. In other aspects of thisembodiment, the endogenous SNAP-25 expressed by cells from anestablished clonal cell line is SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In yet other aspects of thisembodiment, the endogenous SNAP-25 expressed by cells from anestablished clonal cell line is a naturally occurring SNAP-25, such as,e.g., a SNAP-25 isoform or a SNAP-25 subtype. In other aspects of thisembodiment, the endogenous SNAP-25 expressed by cells from anestablished clonal cell line is a naturally occurring SNAP-25 having,e.g., at least 70% amino acid identity, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% amino acid identity with SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ IDNO: 24.

In another embodiment, cells from an established clonal cell line aretransiently or stably engineered to express an exogenous SNAP-25. In anaspect of this embodiment, cells from an established clonal cell lineare transiently or stably engineered to express a naturally-occurringSNAP-25. In other aspects of this embodiment, cells from an establishedclonal cell line are transiently or stably engineered to express thenaturally-occurring SNAP-25 of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In yet other aspects of thisembodiment, cells from an established clonal cell line are transientlyor stably engineered to express a naturally occurring SNAP-25, such as,e.g., a SNAP-25 isoform or a SNAP-25 subtype. In still other aspects ofthis embodiment, cells from an established clonal cell line aretransiently or stably engineered to express a naturally occurringSNAP-25 having, e.g., at least 70% amino acid identity, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% amino acididentity with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, or SEQ ID NO: 24.

In another aspect of the embodiment, cells from an established clonalcell line are transiently or stably engineered to express anon-naturally occurring SNAP-25. In other aspects of this embodiment,cells from an established clonal cell line are transiently or stablyengineered to express a non-naturally occurring SNAP-25 having, e.g., atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95% amino acid identity with SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In other aspects of thisembodiment, cells from an established clonal cell line are transientlyor stably engineered to express a non-naturally occurring SNAP-25having, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 ormore, 40 or more, 50 or more, or 100 or more non-contiguous amino acidsubstitutions, deletions, or additions relative to SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In yetother aspects of this embodiment, cells from an established clonal cellline are transiently or stably engineered to express a non-naturallyoccurring SNAP-25 having, e.g., 1 or more, 2 or more, 3 or more, 4 ormore, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more,20 or more, 30 or more, 40 or more, 50 or more, or 100 or morecontiguous amino acid substitutions, deletions, or additions relative toSEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, orSEQ ID NO: 24.

Assays that detect the cleavage of a BoNT/A substrate after exposure toa BoNT/A can be used to assess whether a cell is expressing anendogenous or an exogenous SNAP-25. In these assays, generation of aSNAP-25 cleavage-product would be detected in cells expressing a SNAP-25after BoNT/A treatment. Non-limiting examples of specific Western blotanalysis, as well as well-characterized reagents, conditions andprotocols are readily available from commercial vendors that include,without limitation, Amersham Biosciences, Piscataway, N.J.; Bio-RadLaboratories, Hercules, Calif.; Pierce Biotechnology, Inc., Rockford,Ill.; Promega Corporation, Madison, Wis., and Stratagene, Inc., LaJolla, Calif. It is understood that these and similar assays for SNAP-25cleavage can be useful in identifying cells expressing an endogenous oran exogenous SNAP-25.

As non-limiting examples, Western blot analysis using an antibody thatrecognizes BoNT/A SNAP-25-cleaved product or both the cleaved anduncleaved forms of SNAP-25 can be used to assay for uptake of BoNT/A.Examples of α-SNAP-25 antibodies useful for these assays include,without limitation, α-SNAP-25 mouse monoclonal antibody SMI-81(Sternberger Monoclonals Inc., Lutherville, Md.), mouse α-SNAP-25monoclonal antibody CI 71.1 (Synaptic Systems, Goettingen, Germany),α-SNAP-25 mouse monoclonal antibody CI 71.2 (Synaptic Systems,Goettingen, Germany), α-SNAP-25 mouse monoclonal antibody SP12 (Abcam,Cambridge, Mass.), α-SNAP-25 rabbit polyclonal antiserum (SynapticSystems, Goettingen, Germany), α-SNAP-25 rabbit polyclonal antiserum(Abcam, Cambridge, Mass.), and α-SNAP-25 rabbit polyclonal antiserumS9684 (Sigma, St Louis, Mo.).

Aspects of the present disclosure comprise, in part, a BoNT/A receptor.As used herein, the term “BoNT/A receptor” refers to either anaturally-occurring BoNT/A receptor or a non-naturally occurring BoNT/Areceptor which preferentially interacts with BoNT/A in a manner thatelicits a BoNT/A intoxication response. As used herein, the term“preferentially interacts” refers to that the equilibrium dissociationconstant (KD) of BoNT/A for a BoNT/A receptor is at least one order ofmagnitude less than that of BoNT/A for any other receptor. Theequilibrium dissociation constant, a specific type of equilibriumconstant that measures the propensity of an BoNT/A-BoNT/A receptorcomplex to separate (dissociate) reversibly into its componentmolecules, namely the BoNT/A and the BoNT/A receptor complex. Thedisassociation constant is defined as KD=[L][R]/[C], where [L] equalsthe molar concentration of BoNT/A, [R] is the molar concentration of aBoNT/A receptor, and [C] is the molar concentration of the BoNT/A-BoNT/Areceptor complex, and where all concentrations are of such componentswhen the system is at equilibrium. The smaller the dissociationconstant, the more tightly bound the BoNT/A is to its receptor, or thehigher the binding affinity between BoNT/A and BoNT/A receptor. Inaspects of this embodiment, the disassociation constant of BoNT/A for aBoNT/A receptor is at least two orders of magnitude less, at least threeorders of magnitude less, at least four orders of magnitude less, or atleast five orders of magnitude less than that of BoNT/A for any otherreceptor. In other aspects of this embodiment, the binding affinity of aBoNT/A that preferentially interacts with a BoNT/A receptor can have anequilibrium disassociation constant of, e.g., of 500 nM or less, 400 nMor less, 300 nM or less, 200 nM, or less 100 nM or less. In otheraspects of this embodiment, the binding affinity of a BoNT/A thatpreferentially interacts with a BoNT/A receptor can have an equilibriumdisassociation constant of, e.g., of 90 nM or less, 80 nM or less, 70 nMor less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20nM, or less 10 nM or less. As used herein, the term “elicits a BoNT/Aintoxication response” refers to the ability of a BoNT/A receptor tointeract with a BoNT/A to form a neurotoxin/receptor complex and thesubsequent internalization of that complex into the cell cytoplasm.

As used herein, the term “naturally occurring BoNT/A receptor” refers toany BoNT/A receptor produced by a naturally-occurring process,including, without limitation, BoNT/A receptor isoforms produced from apost-translational modification, an alternatively-spliced transcript, ora spontaneous mutation, and BoNT/A receptor subtypes. A naturallyoccurring BoNT/A receptor includes, without limitation, a fibroblastgrowth factor receptor 2 (FGFR2), a fibroblast growth factor receptor 3(FGFR3), a synaptic vesicle glycoprotein 2 (SV2), and a complexganglioside like GT1b, such as those described in Ester Fernandez-Salas,et al., Botulinum Toxin Screening Assays, U.S. Patent Publication2008/0003240; Ester Fernandez-Salas, et al., Botulinum Toxin ScreeningAssays, U.S. Patent Publication 2008/0182799; Min Dong et al., SV2 isthe Protein Receptor for Botulinum Neurotoxin A, Science (2006); S.Mahrhold et al, The Synaptic Vesicle Protein 2C Mediates the Uptake ofBotulinum Neurotoxin A into Phrenic Nerves, 580(8) FEBS Lett. 2011-2014(2006), each of which is hereby incorporated by reference in itsentirety. A naturally occurring FGFR2 includes, without limitation, SEQID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63,SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO:68, SEQ ID NO: 69, and SEQ ID NO: 70, or one that substitutes, deletesor adds, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 ormore, 40 or more, 50 or more, or 100 or more amino acids from SEQ ID NO:59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ IDNO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQID NO: 69, and SEQ ID NO: 70. A naturally occurring FGFR3 includes,without limitation, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, orone that substitutes, deletes or adds, e.g., 1 or more, 2 or more, 3 ormore, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more,10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 ormore amino acids from SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27. Anaturally occurring SV2 includes, without limitation, SEQ ID NO: 28, SEQID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, or one that substitutes,deletes or adds, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 ormore, 30 or more, 40 or more, 50 or more, or 100 or more amino acidsfrom SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31.

As used herein, the term “non-naturally occurring BoNT/A receptorvariant” refers to any BoNT/A receptor produced with the aid of humanmanipulation or design, including, without limitation, a BoNT/A receptorproduced by genetic engineering using random mutagenesis or rationaldesign and a BoNT/A receptor produced by chemical synthesis.Non-limiting examples of non-naturally occurring BoNT/A variantsinclude, e.g., conservative BoNT/A receptor variants, non-conservativeBoNT/A receptor variants, BoNT/A receptor chimeric variants and activeBoNT/A receptor fragments.

As used herein, the term “non-naturally occurring BoNT/A receptor”refers to any BoNT/A receptor whose structure was modified with the aidof human manipulation, including, without limitation, a BoNT/A receptorproduced by genetic engineering using random mutagenesis or rationaldesign and a BoNT/A receptor produced by in vitro chemical synthesis.Non-limiting examples of non-naturally occurring BoNT/A receptors aredescribed in, e.g., Ester Fernandez-Salas, et al., Botulinum ToxinScreening Assays, U.S. Patent Publication 2008/0003240; EsterFernandez-Salas, et al., Botulinum Toxin Screening Assays, U.S. PatentPublication 2008/0182799, each of which is hereby incorporated byreference in its entirety. A non-naturally occurring BoNT/A receptor maysubstitute, delete or add, e.g., 1 or more, 2 or more, 3 or more, 4 ormore, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more,20 or more, 30 or more, 40 or more, 50 or more, or 100 or more aminoacids from SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO:60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ IDNO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, orSEQ ID NO: 70.

Thus in an embodiment, a BoNT/A receptor is a naturally occurring BoNT/Areceptor such as, e.g., FGFR2, FGFR3 or SV2. In aspects of thisembodiment, the BoNT/A receptor is a BoNT/A receptor isoform or a BoNT/Areceptor subtype. In aspects of this embodiment, the naturally occurringBoNT/A receptor is the naturally occurring BoNT/A receptor of SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ IDNO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66,SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In otheraspects of this embodiment, the BoNT/A receptor is a naturally occurringBoNT/A receptor having, e.g., at least 70% amino acid identity, at least75%, at least 80%, at least 85%, at least 90%, or at least 95% aminoacid identity with SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64,SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:69, or SEQ ID NO: 70.

In another embodiment, a BoNT/A receptor is a non-naturally occurringBoNT/A receptor, such as, e.g., a genetically-engineered FGFR2, agenetically-engineered FGFR3, or a genetically-engineered SV2. In otheraspects of this embodiment, the BoNT/A receptor is a non-naturallyoccurring BoNT/A receptor having, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% amino acididentity with SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ IDNO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69,or SEQ ID NO: 70. In other aspects of this embodiment, the BoNT/Areceptor is a non-naturally occurring BoNT/A receptor having, e.g., 1 ormore, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more,8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50or more, or 100 or more non-contiguous amino acid substitutions,deletions, or additions relative to SEQ ID NO: 25, SEQ ID NO: 26, SEQ IDNO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63,SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO:68, SEQ ID NO: 69, or SEQ ID NO: 70. In yet other aspects of thisembodiment, the BoNT/A receptor is a non-naturally occurring BoNT/Areceptor having, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 ormore, 30 or more, 40 or more, 50 or more, or 100 or more contiguousamino acid substitutions, deletions, or additions relative to SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ IDNO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66,SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70.

A BoNT/A receptor can be an endogenous BoNT/A receptor or an exogenousBoNT/A receptor. As used herein, the term “endogenous BoNT/A receptor”refers to a BoNT/A receptor naturally present in the cell because it isnaturally encoded within the cell's genome, such that the cellinherently expresses the BoNT/A receptor without the need an externalsource of BoNT/A receptor or an external source of genetic materialencoding a BoNT/A receptor. Expression of an endogenous BoNT/A receptormay be with or without environmental stimulation such as e.g., celldifferentiation or promoter activation. For example, the followingestablished clonal cell lines express at least one endogenous BoNT/Areceptor: BE(2)-M17, Kelly, LA1-55n, N1E-115, N4TG3, N18, Neuro-2a,NG108-15, PC12, SH-SY5Y, SiMa, and SK-N-BE(2)-C. An endogenous BoNT/Areceptor can only be a naturally-occurring BoNT/A receptor ornaturally-occurring variants thereof.

As used herein, the term “exogenous BoNT/A receptor” refers to a BoNT/Areceptor expressed in a cell through the introduction of an externalsource of BoNT/A receptor or an external source of genetic materialencoding a BoNT/A receptor by human manipulation. The expression of anexogenous BoNT/A receptor may be with or without environmentalstimulation such as, e.g., cell differentiation or promoter activation.As a non-limiting example, cells from an established clonal cell linecan express one or more exogenous BoNT/A receptors by transient orstably transfection of a polynucleotide molecule encoding a BoNT/Areceptor, such as, e.g., a FGFR2, a FGFR3, or a SV2. As anothernon-limiting example, cells from an established clonal cell line canexpress one or more exogenous BoNT/A receptors by protein transfectionof the BoNT/A receptors, such as, e.g., a FGFR2, a FGFR3, or a SV2. Anexogenous BoNT/A receptor can be a naturally-occurring BoNT/A receptoror naturally occurring variants thereof, or non-naturally occurringBoNT/A receptor or non-naturally occurring variants thereof.

Thus in an embodiment, cells from an established clonal cell lineexpress an endogenous BoNT/A receptor. In aspects of this embodiment,the endogenous BoNT/A receptor expressed by cells from an establishedclonal cell line is a naturally-occurring BoNT/A receptor. In otheraspects of this embodiment, the endogenous BoNT/A receptor expressed bycells from an established clonal cell line is SEQ ID NO: 25, SEQ ID NO:26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67,SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In yet aspects of thisembodiment, the endogenous BoNT/A receptor expressed by cells from anestablished clonal cell line is a naturally occurring BoNT/A receptor,such as, e.g., a BoNT/A receptor isoform or a BoNT/A receptor subtype.In other aspects of this embodiment, the endogenous BoNT/A receptorexpressed by cells from an established clonal cell line is a naturallyoccurring BoNT/A receptor having, e.g., at least 70% amino acididentity, at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95% amino acid identity with SEQ ID NO: 25, SEQ ID NO: 26, SEQ IDNO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63,SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO:68, SEQ ID NO: 69, or SEQ ID NO: 70.

In another embodiment, cells from an established clonal cell line aretransiently or stably engineered to express an exogenous BoNT/Areceptor. In an aspect of this embodiment, cells from an establishedclonal cell line are transiently or stably engineered to express anaturally-occurring BoNT/A receptor. In other aspects of thisembodiment, cells from an established clonal cell line are transientlyor stably engineered to express the naturally-occurring BoNT/A receptorof SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ IDNO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65,SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ IDNO: 70. In yet other aspects of this embodiment, cells from anestablished clonal cell line are transiently or stably engineered toexpress a naturally occurring BoNT/A receptor, such as, e.g., a BoNT/Areceptor isoform or a BoNT/A receptor subtype. In still other aspects ofthis embodiment, cells from an established clonal cell line aretransiently or stably engineered to express a naturally occurring BoNT/Areceptor having, e.g., at least 70% amino acid identity, at least 75%,at least 80%, at least 85%, at least 90%, or at least 95% amino acididentity with SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ IDNO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69,or SEQ ID NO: 70.

In another aspect of the embodiment, cells from an established clonalcell line are transiently or stably engineered to express anon-naturally occurring BoNT/A receptor. In other aspects of thisembodiment, cells from an established clonal cell line are transientlyor stably engineered to express a non-naturally occurring BoNT/Areceptor having, e.g., at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% amino acid identity with SEQ IDNO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61,SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO:66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. Inother aspects of this embodiment, cells from an established clonal cellline are transiently or stably engineered to express a non-naturallyoccurring BoNT/A receptor having, e.g., 1 or more, 2 or more, 3 or more,4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 ormore, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or morenon-contiguous amino acid substitutions, deletions, or additionsrelative to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO:60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ IDNO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, orSEQ ID NO: 70. In yet other aspects of this embodiment, cells from anestablished clonal cell line are transiently or stably engineered toexpress a non-naturally occurring BoNT/A receptor having, e.g., 1 ormore, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more,8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50or more, or 100 or more contiguous amino acid substitutions, deletions,or additions relative to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ IDNO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQID NO: 69, or SEQ ID NO: 70

In another embodiment, cells from an established clonal cell line aretransiently or stably engineered to express an exogenous FGFR2, anexogenous FGFR3, an exogenous SV2, or any combination thereof. Inaspects of this embodiment, cells from an established clonal cell lineare transiently or stably engineered to express a naturally-occurringFGFR2, a naturally-occurring FGFR3, a naturally-occurring SV2, or anycombination thereof. In yet other aspects of this embodiment, cells froman established clonal cell line are transiently or stably engineered toexpress a non-naturally-occurring FGFR2, a non-naturally-occurringFGFR3, a non-naturally-occurring SV2, or any combination thereof. Instill other aspects of this embodiment, cells from an established clonalcell line are transiently or stably engineered to express either anaturally-occurring FGFR2 or a non-naturally-occurring FGFR2, anaturally-occurring FGFR3 or a non-naturally-occurring FGFR3, anaturally-occurring SV2 or a non-naturally-occurring SV2, or anycombination thereof.

Cells that express one or more endogenous or exogenous BoNT/A receptorscan be identified by routine methods including direct and indirectassays for toxin uptake. Assays that determine BoNT/A binding or uptakeproperties can be used to assess whether a cell is expressing a BoNT/Areceptor. Such assays include, without limitation, cross-linking assaysusing labeled BoNT/A, such as, e.g., [¹²⁵I] BoNT/A, see, e.g., NorikoYokosawa et al., Binding of Clostridium botulinum type C neurotoxin todifferent neuroblastoma cell lines, 57(1) Infect. Immun. 272-277 (1989);Noriko Yokosawa et al., Binding of botulinum type CI, D and Eneurotoxins to neuronal cell lines and synaptosomes, 29(2) Toxicon261-264 (1991); and Tei-ichi Nishiki et al., Identification of proteinreceptor for Clostridium botulinum type B neurotoxin rat brainsynaptosomes, 269(14) J. Biol. Chem. 10498-10503 (1994). Othernon-limiting assays include immunocytochemical assays that detect BoNT/Abinding using labeled or unlabeled antibodies, see, e.g., AtsushiNishikawa et al., The receptor and transporter for internalization ofClostridium botulinum type C progenitor toxin into HT-29 cells, 319(2)Biochem. Biophys. Res. Commun. 327-333 (2004) and immunoprecipitationassays, see, e.g., Yukako Fujinaga et al., Molecular characterization ofbinding subcomponents of Clostridium botulinum type C progenitor toxinfor intestinal epithelial cells and erythrocytes, 150(Pt 5) Microbiology1529-1538 (2004), that detect bound toxin using labeled or unlabeledantibodies. Antibodies useful for these assays include, withoutlimitation, antibodies selected against BoNT/A, antibodies selectedagainst a BoNT/A receptor, such as, e.g., FGFR2, FGFR3, or SV2, and/orantibodies selected against a ganglioside, such as, e.g., GD1a, GD1b,GD3, GQ1b, or GT1b. If the antibody is labeled, the binding of themolecule can be detected by various means, including Western blotanalysis, direct microscopic observation of the cellular location of theantibody, measurement of cell or substrate-bound antibody following awash step, flow cytometry, electrophoresis or capillary electrophoresis,employing techniques well-known to those of skill in the art. If theantibody is unlabeled, one may employ a labeled secondary antibody forindirect detection of the bound molecule, and detection can proceed asfor a labeled antibody. It is understood that these and similar assaysthat determine BoNT/A uptake properties or characteristics can be usefulin identifying cells expressing endogenous or exogenous BoNT/Areceptors.

Assays that monitor the release of a molecule after exposure to BoNT/Acan also be used to assess whether a cell is expressing one or moreendogenous or exogenous BoNT/A receptors. In these assays, inhibition ofthe molecule's release would occur in cells expressing a BoNT/A receptorafter BoNT/A treatment. Well known assays include methods that measureinhibition of radio-labeled catecholamine release from neurons, such as,e.g., [3H] noradrenaline or [3H] dopamine release, see e.g., A Fassio etal., Evidence for calcium-dependent vesicular transmitter releaseinsensitive to tetanus toxin and botulinum toxin type F, 90(3)Neuroscience 893-902 (1999); and Sara Stigliani et al., The sensitivityof catecholamine release to botulinum toxin C1 and E suggests selectivetargeting of vesicles set into the readily releasable pool, 85(2) J.Neurochem. 409-421 (2003), or measures catecholamine release using afluorometric procedure, see, e.g., Anton de Paiva et al., A role for theinterchain disulfide or its participating thiols in the internalizationof botulinum neurotoxin A revealed by a toxin derivative that binds toecto-acceptors and inhibits transmitter release intracellularly, 268(28)J. Biol. Chem. 20838-20844 (1993); Gary W. Lawrence et al., Distinctexocytotic responses of intact and permeabilised chromaffin cells aftercleavage of the 25-kDa synaptosomal-associated protein (SNAP-25) orsynaptobrevin by botulinum toxin A or B, 236(3) Eur. J. Biochem. 877-886(1996); and Patrick Foran et al., Botulinum neurotoxin C1 cleaves bothsyntaxin and SNAP-25 in intact and permeabilized chromaffin cells:correlation with its blockade of catecholamine release, 35(8)Biochemistry 2630-2636 (1996). Other non-limiting examples includeassays that measure inhibition of hormone release from endocrine cells,such as, e.g., anterior pituitary cells or ovarian cells. It isunderstood that these and similar assays for molecule release can beuseful in identifying cells expressing endogenous or exogenous or BoNT/Areceptors.

Assays that detect the cleavage of a BoNT/A substrate after exposure toa BoNT/A can also be used to assess whether a cell is expressing one ormore endogenous or exogenous BoNT/A receptors. In these assays,generation of a BoNT/A substrate cleavage-product, or disappearance ofthe intact BoNT/A substrate, would be detected in cells expressing aBoNT/A receptor after BoNT/A treatment. Non-limiting examples ofspecific Western blot analysis, as well as well-characterized reagents,conditions and protocols are readily available from commercial vendorsthat include, without limitation, Amersham Biosciences, Piscataway,N.J.; Bio-Rad Laboratories, Hercules, Calif.; Pierce Biotechnology,Inc., Rockford, Ill.; Promega Corporation, Madison, Wis., andStratagene, Inc., La Jolla, Calif. It is understood that these andsimilar assays for BoNT/A substrate cleavage can be useful inidentifying cells expressing endogenous or exogenous BoNT/A receptors.

As non-limiting examples, Western blot analysis using an antibody thatrecognizes BoNT/A SNAP-25-cleaved product or both the cleaved anduncleaved forms of SNAP-25 can be used to assay for uptake of BoNT/A.Examples of α-SNAP-25 antibodies useful for these assays include,without limitation, SMI-81α-SNAP-25 mouse monoclonal antibody(Sternberger Monoclonals Inc., Lutherville, Md.), CI 71.1 mouseα-SNAP-25 monoclonal antibody (Synaptic Systems, Goettingen, Germany),CI 71.2 α-SNAP-25 mouse monoclonal antibody (Synaptic Systems,Goettingen, Germany), SP12 α-SNAP-25 mouse monoclonal antibody (Abcam,Cambridge, Mass.), α-SNAP-25 rabbit polyclonal antiserum (SynapticSystems, Goettingen, Germany), α-SNAP-25 rabbit polyclonal antiserumS9684 (Sigma, St. Louis, Mo.), and α-SNAP-25 rabbit polyclonal antiserum(Abcam, Cambridge, Mass.).

Aspects of the present disclosure provide cells that through geneticmanipulation or recombinant engineering are made to express an exogenousSNAP-25 and/or one or more exogenous BoNT/A receptors. Cells useful toexpress an exogenous SNAP-25 and/or one or more exogenous BoNT/Areceptors through genetic manipulation or recombinant engineeringinclude neuronal cells and non-neuronal cells that may or may notexpress an endogenous SNAP-25 and/or one or more endogenous BoNT/Areceptors. It is further understood that such genetically manipulated orrecombinantly engineered cells may express an exogenous SNAP-25 and oneor more exogenous BoNT/A receptors under control of a constitutive,tissue-specific, cell-specific or inducible promoter element, enhancerelement or both. It is understood that any cell is useful as long as thecell can be genetically manipulated or recombinantly engineered toexpresses an exogenous SNAP-25 and/or one or more exogenous BoNT/Areceptors and is capable of undergoing BoNT/A intoxication.

Methods useful for introducing into a cell an exogenous polynucleotidemolecule encoding a component necessary for the cells to undergo theoverall cellular mechanism whereby a BoNT/A proteolytically cleaves aSNAP-25 substrate, such as, e.g., a SNAP-25, a FGFR2, a FGFR3, or a SV2,include, without limitation, chemical-mediated delivery methods, suchas, e.g., calcium phosphate-mediated, diethyl-aminoethyl (DEAE)dextran-mediated, lipid-mediated, polyethyleneimine (PEI)-mediated,polylysine-mediated and polybrene-mediated; physical-mediated deliverymethods, such as, e.g., biolistic particle delivery, microinjection,protoplast fusion and electroporation; and viral-mediated deliverymethods, such as, e.g., retroviral-mediated transfection, see e.g.,Introducing Cloned Genes into Cultured Mammalian Cells, pp. 16.1-16.62(Sambrook & Russell, eds., Molecular Cloning A Laboratory Manual, Vol.3, 3^(rd) ed. 2001); Alessia Colosimo et al., Transfer and Expression ofForeign Genes in Mammalian Cells, 29(2) Biotechniques 314-318, 320-322,324 (2000); Philip Washbourne & A. Kimberley McAllister, Techniques forGene Transfer into Neurons, 12(5) Curr. Opin. Neurobiol. 566-573 (2002);and Current Protocols in Molecular Biology, John Wiley and Sons, pp9.16.4-9.16.11 (2000), each of which is incorporated by reference in itsentirety. One skilled in the art understands that selection of aspecific method to introduce a polynucleotide molecule into a cell willdepend, in part, on whether the cell will transiently or stably containa component necessary for the cells to undergo the overall cellularmechanism whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate.Non-limiting examples of polynucleotide molecule encoding a componentnecessary for the cells to undergo the overall cellular mechanismwhereby a BoNT/A proteolytically cleaves a SNAP-25 substrate as follows:FGFR2 polynucleotide molecule of SEQ ID NO: 130, SEQ ID NO: 131, SEQ IDNO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136,SEQ ID NO: 137, or SEQ ID NO: 138; FGFR3 polynucleotide molecule of SEQID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141; SV2 polynucleotidemolecule of SEQ ID NO: 142, SEQ ID NO: 143, or SEQ ID NO: 144; andSNAP-25 polynucleotide molecule of SEQ ID NO: 145, or SEQ ID NO: 146.

Chemical-mediated delivery methods are well-known to a person ofordinary skill in the art and are described in, e.g., Martin Jordan &Florian Worm, Transfection of Adherent and Suspended Cells by CalciumPhosphate, 33(2) Methods 136-143 (2004); Chun Zhang et al.,Polyethylenimine Strategies for Plasmid Delivery to Brain-Derived Cells,33(2) Methods 144-150 (2004), each of which is hereby incorporated byreference in its entirety. Such chemical-mediated delivery methods canbe prepared by standard procedures and are commercially available, see,e.g., CellPhect Transfection Kit (Amersham Biosciences, Piscataway,N.J.); Mammalian Transfection Kit, Calcium phosphate and DEAE Dextran,(Stratagene, Inc., La Jolla, Calif.); Lipofectamine™ TransfectionReagent (Invitrogen, Inc., Carlsbad, Calif.); ExGen 500 Transfection kit(Fermentas, Inc., Hanover, Md.), and SuperFect and EffecteneTransfection Kits (Qiagen, Inc., Valencia, Calif.).

Physical-mediated delivery methods are well-known to a person ofordinary skill in the art and are described in, e.g., Jeike E. Biewengaet al., Plasmid-Mediated Gene Transfer in Neurons using the BiolisticsTechnique, 71(1) J. Neurosci. Methods. 67-75 (1997); John O'Brien &Sarah C. R. Lummis, Biolistic and Diolistic Transfection: Using the GeneGun to Deliver DNA and Lipophilic Dyes into Mammalian Cells, 33(2)Methods 121-125 (2004); M. Golzio et al., In Vitro and In Vivo ElectricField-Mediated Permeabilization, Gene Transfer, and Expression, 33(2)Methods 126-135 (2004); and Oliver Greschet al., New Non-Viral Methodfor Gene Transfer into Primary Cells, 33(2) Methods 151-163 (2004), eachof which is hereby incorporated by reference in its entirety.

Viral-mediated delivery methods are well-known to a person of ordinaryskill in the art and are described in, e.g., Chooi M. Lai et al.,Adenovirus and Adeno-Associated Virus Vectors, 21(12) DNA Cell Biol.895-913 (2002); Ilya Frolov et al., Alphavirus-Based Expression Vectors:Strategies and Applications, 93(21) Proc. Natl. Acad. Sci. U.S.A.11371-11377 (1996); Roland Wolkowicz et al., Lentiviral Vectors for theDelivery of DNA into Mammalian Cells, 246 Methods Mol. Biol. 391-411(2004); A. Huser & C. Hofmann, Baculovirus Vectors: Novel Mammalian CellGene-Delivery Vehicles and Their Applications, 3(1) Am. J.Pharmacogenomics 53-63 (2003); Tiziana Tonini et al., TransientProduction of Retro viral-and Lentiviral-Based Vectors for theTransduction of Mammalian Cells, 285 Methods Mol. Biol. 141-148 (2004);Manfred Gossen & Hermann Bujard, Tight Control of Gene Expression inEukaryotic Cells by Tetracycline-Responsive Promoters, U.S. Pat. No.5,464,758; Hermann Bujard & Manfred Gossen, Methods for Regulating GeneExpression, U.S. Pat. No. 5,814,618; David S. Hogness, PolynucleotidesEncoding Insect Steroid Hormone Receptor Polypeptides and CellsTransformed With Same, U.S. Pat. No. 5,514,578; David S. Hogness,Polynucleotide Encoding Insect Ecdysone Receptor, U.S. Pat. No.6,245,531; Elisabetta Vegeto et al., Progesterone Receptor Having C.Terminal Hormone Binding Domain Truncations, U.S. Pat. No. 5,364,791;Elisabetta Vegeto et al., Mutated Steroid Hormone Receptors, Methods forTheir Use and Molecular Switch for Gene Therapy, U.S. Pat. No.5,874,534, each of which is hereby incorporated by reference in itsentirety. Such viral-mediated delivery methods can be prepared bystandard procedures and are commercially available, see, e.g.,VIRAPOWER™ Adenoviral Expression System (Invitrogen, Inc., Carlsbad,Calif.) and VIRAPOWER™ Adenoviral Expression System Instruction Manual25-0543 version A, Invitrogen, Inc., (Jul. 15, 2002); and ADEASY™Adenoviral Vector System (Stratagene, Inc., La Jolla, Calif.) andADEASY™ Adenoviral Vector System Instruction Manual 064004f, Stratagene,Inc. Furthermore, such viral delivery systems can be prepared bystandard methods and are commercially available, see, e.g., BD™ Tet-Offand Tet-On Gene Expression Systems (BD Biosciences-Clonetech, Palo Alto,Calif.) and BD™ Tet-Off and Tet-On Gene Expression Systems User Manual,PT3001-1, BD Biosciences Clonetech, (Mar. 14, 2003), GeneSwitch™ System(Invitrogen, Inc., Carlsbad, Calif.) and GENESWITCH™ System AMifepristone-Regulated Expression System for Mammalian Cells version D,25-0313, Invitrogen, Inc., (Nov. 4, 2002); VIRAPOWER™ LentiviralExpression System (Invitrogen, Inc., Carlsbad, Calif.) and VIRAPOWER™Lentiviral Expression System Instruction Manual 25-0501 version E,Invitrogen, Inc., (Dec. 8, 2003); and COMPLETE CONTROL® RetroviralInducible Mammalian Expression System (Stratagene, La Jolla, Calif.) andCOMPLETE CONTROL® Retroviral Inducible Mammalian Expression SystemInstruction Manual, 064005e.

As mentioned above, an exogenous component necessary for the cells toundergo the overall cellular mechanism whereby a BoNT/A proteolyticallycleaves a SNAP-25 substrate, such as, e.g., a SNAP-25, a FGFR2, a FGFR3,or a SV2 disclosed in the present specification can be introduced into acell. Any and all methods useful for introducing such an exogenouscomponent with a delivery agent into a cell population can be usefulwith the proviso that this method introduce the exogenous componentdisclosed in the present specification in at least 50% of the cellswithin a given cell population. Thus, aspects of this embodiment caninclude a cell population in which, e.g., at least 50%, at least 60%, atleast 70%, at least 80%, or at least 90% of the given cell populationcontains an exogenous component necessary for the cells to undergo theoverall cellular mechanism whereby a BoNT/A proteolytically cleaves aSNAP-25 substrate, such as, e.g., a SNAP-25, a FGFR2, a FGFR3, or a SV2disclosed in the present specification. As used herein, the term“delivery agent” refers to any molecule that enables or enhancesinternalization of a covalently-linked, non-covalently-linked or in anyother manner associated with a polypeptide into a cell. Thus, the term“delivery agent” encompasses, without limitation, proteins, peptides,peptidomimetics, small molecules, polynucleotide molecules, liposomes,lipids, viruses, retroviruses and cells that, without limitation,transport a covalently or non-covalently linked molecule to the cellmembrane, cell cytoplasm or nucleus. It further is understood that theterm “delivery agent” encompasses molecules that are internalized by anymechanism, including delivery agents which function via receptormediated endocytosis and those which are independent of receptormediated endocytosis.

A delivery agent can also be an agent that enables or enhances cellularuptake of a covalently linked component, like FGFR2, FGFR3, SV2, orSNAP-25, such as, e.g., by chemical conjugation or by geneticallyproduced fusion proteins. Methods that covalently link delivery agentsand methods of using such agents are described in, e.g., Steven F.Dowdy, Protein Transduction System and Methods of Use Thereof,International Publication No WO 00/34308; Gérard Chassaing & AlainProchiantz, Peptides which can be Used as Vectors for the IntracellularAddressing of Active Molecules, U.S. Pat. No. 6,080,724; Alan Frankel etal., Fusion Protein Comprising TAT-derived Transport Moiert, U.S. Pat.No. 5,674,980; Alan Frankel et al., TAT-derived Transport PolypeptideConjugates, U.S. Pat. No. 5,747,641; Alan Frankel et al., TAT-derivedTransport Polypeptides and Fusion Proteins, U.S. Pat. No. 5,804,604;Peter F. J. O'Hare et al., Use of Transport Proteins, U.S. Pat. No.6,734,167; Yao-Zhong Lin & Jack J. Hawiger, Method for ImportingBiologically Active Molecules into Cells, U.S. Pat. No. 5,807,746;Yao-Zhong Lin & Jack J. Hawiger, Method for Importing BiologicallyActive Molecules into Cells, U.S. Pat. No. 6,043,339; Yao-Zhong Lin etal., Sequence and Method for Genetic Engineering of Proteins with CellMembrane Translocating Activity, U.S. Pat. No. 6,248,558; Yao-Zhong Linet al., Sequence and Method for Genetic Engineering of Proteins withCell Membrane Translocating Activity, U.S. Pat. No. 6,432,680; Jack J.Hawiger et al., Method for Importing Biologically Active Molecules intoCells, U.S. Pat. No. 6,495,518; Yao-Zhong Lin et al., Sequence andMethod for Genetic Engineering of Proteins with Cell MembraneTranslocating Activity, U.S. Pat. No. 6,780,843; Jonathan B. Rothbard &Paul A Wender, Method and Composition for Enhancing Transport AcrossBiological Membranes, U.S. Pat. No. 6,306,993; Jonathan B. Rothbard &Paul A Wender, Method and Composition for Enhancing Transport AcrossBiological Membranes, U.S. Pat. No. 6,495,663; and Pamela B. Davis etal., Fusion Proteins for Protein Delivery, U.S. Pat. No. 6,287,817, eachof which is incorporated by reference in its entirety.

A delivery agent can also be an agent that enables or enhances cellularuptake of a non-covalently associated component, like FGFR2, FGFR3,SV2C, or SNAP-25. Methods that function in the absence of covalentlinkage and methods of using such agents are described in, e.g., GillesDivita et al, Peptide-Mediated Transfection Agents and Methods of Use,U.S. Pat. No. 6,841,535; Philip L Felgner and Olivier Zelphati,Intracellular Protein Delivery Compositions and Methods of Use, U.S.Patent Publication No. 2003/0008813; and Michael Karas, IntracellularDelivery of Small Molecules, Proteins and Nucleic Acids, U.S. PatentPublication 2004/0209797, each of which is incorporated by reference inits entirety. Such peptide delivery agents can be prepared and used bystandard methods and are commercially available, see, e.g. the CHARIOT™Reagent (Active Motif, Carlsbad, Calif.); BIO-PORTER® Reagent (GeneTherapy Systems, Inc., San Diego, Calif.), BIO TREK™ Protein DeliveryReagent (Stratagene, La Jolla, Calif.), and PRO-JECT™ ProteinTransfection Reagent (Pierce Biotechnology Inc., Rockford, Ill.).

Aspects of the present disclosure can also be described as follows:

-   1. An established clonal cell line comprising a cell susceptible to    intoxication by BoNT/A.-   2. The established clonal cell line of 1, wherein the clonal cell    line is selected from a parental SiMa cell line.-   3. The established clonal cell line of 1 or 2, wherein the cell is    susceptible to BoNT/A intoxication by about 100 pM or less, by about    50 pM or less, by about 10 pM or less, by about 5 pM or less, by    about 1 pM or less, by about 0.5 pM or less, or by about 0.1 pM or    less of a BoNT/A.-   4. The established clonal cell line of 1 or 2, wherein    susceptibility to BoNT/A intoxication is measured before    differentiation of the cell.-   5. The established clonal cell line of 2, wherein the    undifferentiated state, the cell exhibits at least a 1.5-fold    increase in gene expression of one or more genes relative to the    expression of the one or more genes in a cell from a 2D6 cell line,    the genes being taken from Table 5, or wherein the cell exhibits at    least a 1.5-fold decrease in gene expression of one or more genes    relative to the expression of the one or more genes in a cell from a    2D6 cell line, the genes being taken from Table 6.-   6. The established clonal cell line of 2, wherein the differentiated    state, the cell exhibits at least a 1.5-fold increase in gene    expression of one or more genes relative to the expression of the    one or more genes in a cell from a 2D6 cell line, the genes being    taken from Table 9, or wherein the cell exhibits at least a 1.5-fold    decrease in gene expression of one or more genes relative to the    expression of the one or more genes in a cell from a 2D6 cell line,    the genes being taken from Table 10.-   7. The established clonal cell line of 2, wherein the    undifferentiated state, the cell exhibits at least a 1.5-fold    increase in gene expression of one or more genes relative to the    expression of the one or more genes in a cell from a parental SiMa    cell line, the genes being taken from Table 7, or wherein the cell    exhibits at least a 1.5-fold decrease in gene expression of one or    more genes relative to the expression of the one or more genes in a    cell from a parental SiMa cell line, the genes being taken from    Table 8.-   8. The established clonal cell line of 2, wherein the differentiated    state, the cell exhibits at least a 1.5-fold increase in gene    expression of one or more genes relative to the expression of the    one or more genes in a cell from a parental SiMa cell line, the    genes being taken from Table 11, or wherein the cell exhibits at    least a 1.5-fold decrease in gene expression of one or more genes    relative to the expression of the one or more genes in a cell from a    parental SiMa cell line, the genes being taken from Table 12.-   9. The established clonal cell line of 1 or 2, wherein the cell    exhibits a sensitivity for BoNT/A activity that is 100 pM or less or    about 25 pM for about 5 or more cell passages, 10 or more cell    passages, 15 or more cell passages, 20 or more cell passages, 25 or    more cell passages, 30 or more cell passages, 35 or more cell    passages, 40 or more cell passages, 45 or more cell passages, 50 or    more cell passage, 55 or more cell passage, or 60 or more cell    passage.-   10. The established clonal cell line of 1 or 2, wherein the cell    from an established clonal cell line exhibits a well defined signal    to noise ratio for the upper asymptote for BoNT/A activity of at    least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1,    at least 8:1, at least 9:1, at least 10:1, over, e.g., 5 or more    cell passages, 10 or more cell passages, 15 or more cell passages,    20 or more cell passages, 25 or more cell passages, 30 or more cell    passages, 35 or more cell passages, 40 or more cell passages, 45 or    more cell passages, 50 or more cell passage, 55 or more cell    passage, or 60 or more cell passage.-   11. An established clonal cell line comprising a cell susceptible to    intoxication by BoNT/A produced from a parental SiMa cell line,    wherein the cell is susceptible to BoNT/A intoxication by about 100    pM or less, by about 50 pM or less, by about 10 pM or less, by about    5 pM or less, by about 1 pM or less, by about 0.5 pM or less, or by    about 0.1 pM or less of a BoNT/A.-   12. An established clonal cell line comprising cells susceptible to    intoxication by BoNT/A,    -   wherein the clonal cell line is selected from a parental SiMa        cell line; and    -   wherein the clonal cell line comprises cells susceptible to        BoNT/A intoxication by about 100 pM or less of a BoNT/A.-   13. The established clonal cell line of 12, wherein the parental    SiMa cell line is the parental SiMa cell line DSMZ ACC 164.-   14. The established clonal cell line of 12, wherein the cell    exhibits a sensitivity for BoNT/A activity that is 100 pM or less or    about 25 pM for about 5 or more cell passages, 10 or more cell    passages, 15 or more cell passages, 20 or more cell passages, 25 or    more cell passages, 30 or more cell passages, 35 or more cell    passages, 40 or more cell passages, 45 or more cell passages, 50 or    more cell passage, 55 or more cell passage, or 60 or more cell    passage.-   15. The established clonal cell line of 12, wherein the cell from an    established clonal cell line exhibits a well defined signal to noise    ratio for the upper asymptote for BoNT/A activity of at least 3:1,    at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least    8:1, at least 9:1, at least 10:1, over, e.g., 5 or more cell    passages, 10 or more cell passages, 15 or more cell passages, 20 or    more cell passages, 25 or more cell passages, 30 or more cell    passages, 35 or more cell passages, 40 or more cell passages, 45 or    more cell passages, 50 or more cell passage, 55 or more cell    passage, or 60 or more cell passage.-   16. The established clonal cell line of 12, wherein susceptibility    to BoNT/A intoxication is measured before differentiation of the    cell or after differentiation of the cell.-   17. An established clonal cell line comprising cells susceptible to    intoxication by BoNT/A,    -   wherein the cells exhibits at least a 1.5-fold difference in        gene expression levels of three or more genes as compared to the        expression levels of these genes in cells from the 2D6 cell        line;    -   wherein the gene expression levels are selected from th group        ADAMTS9, ASCL1, BASP1, DOK5, DPYD, GNB4, GNG11, GTSF1, MAOA,        MINA, MSN, PEG3, PLK2, PRSS12, RNF182, SLC44A5, SPARC, TFPI2,        and TPTE; and    -   wherein the clonal cell line comprises cells susceptible to        BoNT/A intoxication by about 100 pM or less of a BoNT/A.-   18. The established clonal cell line of 17, wherein the cells    exhibits at least a 2.0-fold difference in gene expression levels of    one or more genes as compared to the expression levels of these    genes in cells from the 2D6 cell line, at least a 3.0-fold    difference in gene expression levels of one or more genes as    compared to the expression levels of these genes in cells from the    2D6 cell line, or at least a 4.0-fold difference in gene expression    levels of one or more genes as compared to the expression levels of    these genes in cells from the 2D6 cell line.-   19. The established clonal cell line of 17, wherein the gene    expression levels are measured before differentiation of the cell or    after differentiation of the cell.-   20. The established clonal cell line of 17, wherein the cell    exhibits a sensitivity for BoNT/A activity that is 100 pM or less or    about 25 pM for about 5 or more cell passages, 10 or more cell    passages, 15 or more cell passages, 20 or more cell passages, 25 or    more cell passages, 30 or more cell passages, 35 or more cell    passages, 40 or more cell passages, 45 or more cell passages, 50 or    more cell passage, 55 or more cell passage, or 60 or more cell    passage.-   21. The established clonal cell line of 17, wherein the cell from an    established clonal cell line exhibits a well defined signal to noise    ratio for the upper asymptote for BoNT/A activity of at least 3:1,    at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least    8:1, at least 9:1, at least 10:1, over, e.g., 5 or more cell    passages, 10 or more cell passages, 15 or more cell passages, 20 or    more cell passages, 25 or more cell passages, 30 or more cell    passages, 35 or more cell passages, 40 or more cell passages, 45 or    more cell passages, 50 or more cell passage, 55 or more cell    passage, or 60 or more cell passage.

EXAMPLES Example I Screening for Cell Lines Comprising Cells Susceptibleto BoNT/A Intoxication

The following example illustrates how to identify clonal cells from aparental established cell line that are susceptible to BoNT/Aintoxication or have neurotoxin uptake capacity.

1. Isolation of Clonal Cell Lines.

Companion patent application Ester Fernandez-Salas, et al., Immuno-BasedBotulinum Toxin Serotype A Activity Assays, U.S. patent application Ser.No. 12/403,531 identified several established cell lines useful forconducting the disclosed immuno-based methods of detecting BoNT/Aactivity, including, e.g., LA1-55n (ECACC 06041203), N18 (ECACC88112301), Neuro-2a (ATCC CCL-131), PC12 (ATCC CRL-1721), SH-SY5Y (ATCCCRL-2266), and SiMa (DSMZ ACC 164). During characterization of the SiMacell line, it was discovered that the cells comprising this establishedcell line comprised at least five different cellular phenotypes. Todetermine whether any one of these phenotypically-distinct cell typeswas responsible for the susceptibility of this cell line to BoNT/Aintoxication, two different limited-dilution screens were conducted toobtain single colony isolates for each phenotypically-distinct celltype.

In both screens, a suitable density of cells from a SiMa stock weregrown in RPMI 1640 medium, having 10% fetal bovine serum, 1%Penicillin-Streptomycin, 2 mM L-Glutamine, contained in a T175 CollagenIV coated flask. After the second passage, the cells weretrypsin-treated to produce a cell suspension and the cell concentrationwas determined. About 4.0×10⁶ cells from this cell suspension wastransferred into a 50 mL tube and the cells were dissociated into singlecells by repeated vigorous expulsion through an 18.5 gauge needle usinga 10 mL syringe. Cells from this disassociated single-cell suspensionwere then diluted to a concentration of 0.2×10⁶ cells/mL by adding 15 mLof fresh growth medium, and 2.5 μL of this dilution was added to 50 mLof fresh growth medium to obtain a concentration of 10 cells/mL. Fromthis final dilution stock, 100 μL of growth medium was added to eachwell of a 96-well Collagen IV coated plates (final average density ofone cell/well), and the cells were grown undisturbed in a 37° C.incubator under 5% carbon dioxide for four weeks. A total of nineteen96-well plates were setup for analysis. Plates were microscopicallyexamined periodically to assess colony growth. After four weeks, eachwell was microscopically examined to identify growing colonies, and foreach growing colony identified, 100 μL of fresh growth medium was addedto each well and the cells were grown undisturbed in a 37° C. incubatorunder 5% carbon dioxide for two weeks. After two additional weeks ofgrowth, the growing single colonies were trypsin-treated and transferredto a new 96-well plate for continued growth. Once colonies grew to about1,000 cells, based on visual inspection, the cells were trypsin-treatedand each cell-suspension was transferred into a new well from a 24-wellCollagen IV-coated plate. The cells were grown in a 37° C. incubatorunder 5% carbon dioxide with fresh growth medium being replenished every2-3 days, if needed. The cells were grown until the culture reachedapproximately 60% confluence or greater, at which point the cells weretrypsin-treated and each cell-suspension was transferred into a 25 cm²Collagen IV-coated flask. The cells were grown in a 37° C. incubatorunder 5% carbon dioxide with fresh growth medium being replenished every2-3 days, if needed. Once the cells in the flask reached 70-80%confluence, they were frozen and stored in liquid nitrogen until theclonal cell lines were tested to determine their susceptibility toBoNT/A intoxication. Of the 1,824 colony isolates initially setup fromboth screens, 130 clonal cell lines were selected based on viability andgrowth criteria and expanded for subsequent screening procedures.

2. Primary Screen for BoNT/A Intoxication Susceptibility of Cells from aClonal Cell Line Using a BoNT/A Complex.

One way to determine whether cells from a clonal cell line weresusceptible to BoNT/A intoxication was to conduct a primary screen usinga CELLTITER-GLO® luminescent cell viability assay (Promega, Madison,Wis.) for normalizing the cell number in each well and an immuno-basedmethod for determining BoNT/A activity as described in EsterFernandez-Salas, et al., Immuno-Based Botulinum Toxin Serotype AActivity Assays, U.S. patent application Ser. No. 12/403,531, which ishereby incorporated by reference in its entirety. Two separate 96-wellplates were set up, one for the BoNT/A activity assay and the other forthe cell viability assay.

To conduct the BoNT/A activity assay, a lysate was prepared from cellstreated with BoNT/A by plating cells from each isolated clonal cell lineinto a well from a poly-D-lysine 96-well plate (BD Biosciences, Bedford,Mass.). The wells contained 0.1 mL of a serum-free medium comprisingMinimum Essential Medium, 2 mM GlutaMAX™ I with Earle's salts, 1× B27supplement, 1×N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mMHEPES and 25 μg/mL GT1b (302-0.1-M005, Alexis Biochemicals, San Diego,Calif.). These cells were incubated in a 37° C. incubator under 5%carbon dioxide until the cells differentiated, as assessed by standardand routine morphological criteria, such as growth arrest and neuriteextension (approximately 3 days). The media from the differentiatedcells was aspirated from each well and replaced with fresh mediacontaining 1 nM of a BoNT/A complex. After a 24 hrs incubation, thecells were washed by aspirating the growth media and rinsing each wellwith 200 μL of 1×PBS. To harvest the cells, 1×PBS was aspirated, thecells lysed by adding 30 μl of Lysis Buffer comprising 20 mM Tris-HCl(pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 to eachwell, and the plate incubated on a shaker rotating at 500 rpm for 30minutes at 4° C. The plate was centrifuged at 4000 rpm for 20 minutes at4° C. to pellet cellular debris and the supernatant was transferred to acapture antibody coated 96-well plate to perform the detection step.

To prepare an α-SNAP-25 capture antibody solution, the α-SNAP-25 mousemonoclonal antibody contained in the ascites from hybridoma cell line2E2A6 (Example VII) was purified using a standard Protein A purificationprotocol.

To prepare an α-SNAP-25 detection antibody solution, α-SNAP-25 rabbitpolyclonal antibody S9684 (Sigma, St. Louis, Mo.) was conjugated toRuthenium(II)-tris-bipyridine-(4-methysulfonate) NHS ester labelingreagent (Meso Scale Discovery, Gaithersburg, Md.) according to themanufacturer's instructions (Meso Scale Discovery, Gaithersburg, Md.).The conjugation reaction was performed by adding 30 μL of distilledwater reconstituted MSD SULFO-TAG™ stock solution to 200 μL of 2 mg/mLα-SNAP-25 polyclonal antibodies and incubating the reaction at roomtemperature for 2 hours in the dark. The labeled antibodies werepurified using a standard spin column protocol and the proteinconcentration determined using a standard colorimetric protein assay.The absorbance of the α-SNAP-25 antibody/MSD SULFO-TAG™ conjugate wasmeasured at 455 nm using a spectrophotometer to determine theconcentration in moles per liter. The detection antibody solution wasstored at 4° C. until needed.

To prepare an α-SNAP-25 solid phase support comprising an α-SNAP-25capture antibody, approximately 5 μL of the appropriate α-SNAP-25monoclonal antibody solution (20 μg/mL in 1×PBS) is added to each wellof a 96-well MSD High Bind plate and the solution is allowed to air dryin a biological safety cabinet for 2-3 hours in order to liquidevaporate the solution. The capture antibody-bound wells were sealed andstored at 4° C. until needed.

To detect the presence of a cleaved SNAP-25 product by ECL sandwichELISA, the wells from an α-SNAP-25 solid phase support were then blockedby adding 150 μL of Blocking Buffer comprising 2% Amersham BlockingReagent (GE Life Sciences, Piscataway, N.J.) and 10% goat serum (VWR,West Chester, Pa.) at room temperature for 2 hours. The Blocking Bufferwas aspirated, 25 μL of a lysate from cells treated with BoNT/A wasadded to each well and the plates were incubated at 4° C. for overnight.Plate wells were washed three times by aspirating the cell lysate andrinsing each well three times with 200 μL 1×PBS, 0.1% TWEEN-20®(polyoxyethylene (20) sorbitan monolaureate). After washing, 25 μl of 5μg/mL α-SNAP-25 detection antibody solution comprising 2% AmershamBlocking Reagent in 1×PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitanmonolaureate) was added to each well, sealed, and incubated at roomtemperature for 1 hour with shaking. After α-SNAP-25 detection antibodyincubation, the wells were washed three times with 200 μL 1×PBS, 0.1%TWEEN-20® (polyoxyethylene (20) sorbitan monolaureate). After washing150 μL of 1× Read Buffer (Meso Scale Discovery, Gaithersburg, Md.) wasadded to each well and the plates were read using a SECTOR™ Imager 6000Image Reader (Meso Scale Discovery, Gaithersburg, Md.). The raw data wascollected using the ECL imager.

To conduct the luminescent cell viability assay, cells from eachisolated clonal cell line were plated and differentiated as describedabove. The media from the differentiated cells was aspirated from eachwell and replaced with 100 μl of the reagent mixture comprising theCELLTITER-GLO® substrate and CELLTITER-GLO® buffer according to themanufacturer's instructions. The plate was then protected from light andmixed for two minutes on an orbital shaker. The solution from the96-well cell plate with clear bottom was transferred to a whiteluminometer plate and after 10 minutes incubation was read on theluminometer.

These results indicated that the cell viability readings ranged from9,838 to 10,423,188 and BoNT/A activity signals ranged from 84 to 27,242(Table 1). In addition, a ratio of BoNT/A activity over cell viabilitywas calculated to normalize the data, although there was not always a1:1 ratio of viability and activity. Seven clonal cell lines exhibitedactivity/viability ratios of about 0.2×10⁻² or greater: H1, A7, A11,F10, A9, A10, and C11 (Table 1). Nine clonal cell lines exhibitedactivity/viability ratios of about 0.125×10⁻² to about 0.150×10⁻²: H3,H8, E11, H10, A6, C5, C12, D11, and B5 (Table 1). Two cell linesexhibited activity/viability ratios of about 0.100×10⁻²: D7 and B10(Table 1). Based on these activity/viability ratios in conjunction withthe cell viability and BoNT/A activity signal measurements, these 18clonal cell lines were selected for further testing by conducting asecondary screen using a BoNT/A activity assay in order to generate adose-response curve to BoNT/A.

TABLE 1 Primary Screen for Clonal Cell Lines Susceptible to BoNT/AIntoxication Using Viability and Activity Signals Cell Viability BoNT/AReading Activity Activity/Viability Well (RLU) Signal (RLU) Ratio A12634417 2425 0.092 × 10⁻² B1 731808 198 0.027 × 10⁻² C1 4258160 16410.039 × 10⁻² D1 6554606 4885 0.075 × 10⁻² E1 2374483 897 0.038 × 10⁻² F12801444 1842 0.066 × 10⁻² G1 5094510 4579 0.090 × 10⁻² H1 3808699 108610.285 × 10⁻² A2 9593106 7090 0.074 × 10⁻² B2 5374966 5673 0.106 × 10⁻²C2 4170158 2972 0.071 × 10⁻² D2 31283 88 0.281 × 10⁻² E2 5706902 37430.066 × 10⁻² F2 4884798 4568 0.094 × 10⁻² G2 3552045 2190 0.062 × 10⁻²H2 3743690 3421 0.091 × 10⁻² A3 15372 89 0.579 × 10⁻² B3 5381672 15510.029 × 10⁻² C3 4270534 3959 0.093 × 10⁻² D3 48702 97 0.199 × 10⁻² E34707349 2671 0.057 × 10⁻² F3 3053132 1539 0.050 × 10⁻² G3 5518292 41950.076 × 10⁻² H3 4156632 6339 0.153 × 10⁻² A4 19793 89 0.450 × 10⁻² B46224766 4299 0.069 × 10⁻² C4 3212859 3202 0.100 × 10⁻² D4 4376180 23670.054 × 10⁻² E4 3931793 3975 0.101 × 10⁻² F4 1150134 526 0.046 × 10⁻² G419023 94 0.494 × 10⁻² H4 3368425 2645 0.079 × 10⁻² A5 8231689 7809 0.095× 10⁻² B5 5332004 6545 0.123 × 10⁻² C5 3869441 5042 0.130 × 10⁻² D52554300 1161 0.045 × 10⁻² E5 3781403 764 0.020 × 10⁻² F5 4608801 37200.081 × 10⁻² G5 7105403 7330 0.103 × 10⁻² H5 5585607 7544 0.135 × 10⁻²A6 7698708 10152 0.132 × 10⁻² B6 4706560 3196 0.068 × 10⁻² C6 58662516145 0.105 × 10⁻² D6 2285787 768 0.034 × 10⁻² E6 2471979 1038 0.042 ×10⁻² F6 10958 91 0.830 × 10⁻² G6 19590 91 0.465 × 10⁻² H6 2057687 25640.125 × 10⁻² A7 10132753 27242 0.269 × 10⁻² B7 2977371 1642 0.055 × 10⁻²C7 5746577 4878 0.085 × 10⁻² D7 5861338 6044 0.103 × 10⁻² E7 28199101399 0.050 × 10⁻² F7 23523 84 0.357 × 10⁻² G7 6232344 4031 0.065 × 10⁻²H7 4916756 2759 0.056 × 10⁻² A8 5945833 5611 0.094 × 10⁻² B8 45910022128 0.046 × 10⁻² C8 5648241 8758 0.155 × 10⁻² D8 4207887 3360 0.080 ×10⁻² E8 5894699 3844 0.065 × 10⁻² F8 5585487 4651 0.083 × 10⁻² G84758800 4545 0.096 × 10⁻² H8 5439137 8017 0.147 × 10⁻² A9 10423188 207060.199 × 10⁻² B9 6493893 4444 0.068 × 10⁻² C9 5742047 5788 0.101 × 10⁻²D9 3424405 1848 0.054 × 10⁻² E9 5771943 8064 0.140 × 10⁻² F9 24524 880.359 × 10⁻² G9 7291510 6674 0.092 × 10⁻² H9 4169883 6048 0.145 × 10⁻²A10 9050811 17617 0.195 × 10⁻² B10 5590113 5175 0.093 × 10⁻² C10 23258071713 0.074 × 10⁻² D10 2644116 2589 0.098 × 10⁻² E10 4210945 2666 0.063 ×10⁻² F10 2932749 6390 0.218 × 10⁻² G10 3980127 3114 0.078 × 10⁻² H104519035 6187 0.137 × 10⁻² A11 9870025 24690 0.250 × 10⁻² B11 49738922235 0.045 × 10⁻² C11 3326448 6405 0.193 × 10⁻² D11 5747961 7169 0.125 ×10⁻² E11 5831546 8432 0.145 × 10⁻² F11 5291803 4317 0.082 × 10⁻² G113499587 2790 0.080 × 10⁻² H11 3225750 4518 0.140 × 10⁻² A12 9838 890.905 × 10⁻² B12 3728293 2016 0.054 × 10⁻² C12 4086862 5308 0.130 × 10⁻²D12 5044526 3860 0.077 × 10⁻² E12 4787913 2719 0.057 × 10⁻² F12 51016952585 0.051 × 10⁻² G12 2887390 1622 0.056 × 10⁻² H12 2829307 5105 0.180 ×10⁻²

To conduct BoNT/A activity assay in order to generate a dose-responsecurve to BoNT/A, cells from each isolated clonal cell line were seededin a poly-D-lysine 96-well plate and differentiated as described above.Of the top 18 clonal cell lines selected for further testing, only 8remained viable, the remaining 10 clonal cell lines failed to regrow andwere lost. The media from the differentiated cells was aspirated fromeach well and replaced with fresh media containing either 0 (untreatedsample), 0.03 pM, 0.1 pM, 0.3 pM, 0.9 pM, 2.8 pM, 8.3 pM, and 25 pM of aBoNT/A complex. After a 24 hrs treatment, the cells were washed,incubated for an additional two days in serum-free medium without toxinto allow for the cleavage of the SNAP-25 substrate, and harvested asdescribed above. The supernatant was transferred to a capture antibodycoated 96-well plate to perform the detection step.

The α-SNAP-25 capture antibody solution, the α-SNAP-25 detectionantibody solution, and the α-SNAP-25 solid phase support were preparedas described above. Detection of the presence of cleaved SNAP-25 productby ECL sandwich ELISA analysis was performed as described above. The rawdata obtained from the ECL imager was then transferred to SigmaPlot v.9.0 and a 4-parameter logistics fit was used to define the dose-responsecurves. There were no constraints used for the 4-parameter logisticfunction when plotting the data. Graphical reports were generated usingthe following analysis: R2 (correlation coefficient), a (Max for dataset), b (hillslope), and X0±SE (EC₅₀ value±standard error).

These results indicate that of the 8 clonal cell lines tested, only twowere more susceptible to BoNT/A intoxication when compared to cellscomprising the parental SiMa cell line (Table 2). For example, cellscomprising the parental SiMa cell line exhibited an EC₅₀ activity forBoNT/A of 2.20 pM, whereas cells comprising the H1 and A10 cell linesexhibited an EC₅₀ activity for BoNT/A of 1.76 pM or less. Conversely,cells comprising the H10 and D11 cell line exhibited an EC₅₀ activityfor BoNT/A that was higher than that observed for cells comprising theparental SiMa cell line, but less than 5.0 pM. It should be noted thatany clonal cell line exhibiting an EC₅₀ activity for BoNT/A that is 5.0pM or less (100 U/mL or less) is useful for an immuno-based method fordetermining BoNT/A activity described in Ester Fernandez-Salas, et al.,Immuno-Based Botulinum Toxin Serotype A Activity Assays, U.S. patentapplication Ser. No. 12/403,531.

TABLE 2 Secondary Screen for Clonal Cell Lines Susceptible to BoNT/AIntoxication BoNT/A Activity Clone EC₅₀ (pM) Signal_(MIN) (RLU)Signal_(MAX) (RLU) SiMa 2.20 123  47,611 H1 0.77 193 125,252 H3 ND ND NDB5 >25.0   115  22,277 C5 ND ND ND A6 ND ND ND A7 5.59 153 100,412 D7 NDND ND H8 ND ND ND A9 ND ND ND A10 1.76 207 113,029 B10 6.35 147 101,935F10 >25.0   111  32,663 H10 2.80 171 120,975 A11 ND ND ND C11 ND ND NDD11 4.4     1.84 105,527 E11 ND ND ND C12 ND ND ND ND = Not determined.3. Primary Screen for BoNT/A Intoxication Susceptibility of Cells from aClonal Cell Line Using a Formulated BoNT/A Pharmaceutical Product.

Another way to determine whether cells from a clonal cell line weresusceptibility to BoNT/A intoxication was to conduct a primary screen bygenerating a dose-response curve to BoNT/A and calculate the EC₅₀ valueusing an immuno-based method for determining BoNT/A activity asdescribed in Ester Fernandez-Salas, et al., Immuno-Based Botulinum ToxinSerotype A Activity Assays, U.S. patent application Ser. No. 12/403,531,which is hereby incorporated by reference in its entirety.

Initially six of 35 clonal cell line isolates were examined using a fulldose response to BoNT/A. To prepare a lysate from cells treated withBoNT/A, cells from six clonal cell line were seeded in a poly-D-lysine96-well plate and differentiated as described above in Section 2 exceptthat the serum-free medium comprised 60 μg/mL GT1b. The clonal celllines used were 3D8, YB8, 1D4, 2D6, 1E11, 2F5. The H1 and H10 clonalcell lines identified above, along with the parental SiMa cell line,were used as references to identify cell lines highly susceptible toBoNT/A intoxication. The media from the differentiated cells wasaspirated from each well and replaced with fresh media containing either0 (untreated sample), 0.98 U/mL, 1.9 U/mL, 3.91 U/mL, 7.81 U/mL, 15.6U/mL, 31.3 U/mL, 62.5 U/mL, 125 U/mL, 250 U/mL, or 500 U/mL of aformulated BoNT/A pharmaceutical product. After a 24 hrs treatment, thecells were washed, incubated for an additional two days in serum-freemedium without toxin to allow for the cleavage of the SNAP-25 substrate.To harvest the cells, 1×PBS was aspirated, the cells lysed by adding 30μl of Lysis Buffer comprising 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mMEDTA, 1 mM EGTA, 1% Triton X-100 to each well, and the plate incubatedon a shaker rotating at 500 rpm for 30 minutes at 4° C. The plate wascentrifuged at 4000 rpm for 20 minutes at 4° C. to pellet cellulardebris. The protein concentration was determined using standard methods.The supernatant was transferred to a capture antibody coated 96-wellplate to perform the detection step of the immuno-based method.

The α-SNAP-25 capture antibody solution, the α-SNAP-25 detectionantibody solution, and the α-SNAP-25 solid phase support were preparedas described above in Section 2. Detection of the presence of SNAP-25cleavage product by ECL sandwich ELISA analysis was performed, collecteddata was analyzed and the EC₅₀ calculated as described above in Section2, except that SigmaPlot v. 10 was used.

These results show that clonal cell line H1 comprises cells that aremore susceptible to BoNT/A than the parental SIMa cell line (Table 3).In addition, results indicate that although clonal cell lines H10, 1D4,1E11, 2D6, 2F5, 3D8, and YB8 all comprise cells that are lesssusceptible to BoNT/A than the parental SiMa cell line, all have an EC₅₀of BoNT/A activity that is below 30 U/mL (Table 3). It should be notedthat any clonal cell line exhibiting an EC₅₀ activity for BoNT/A that is100 U/mL or less (5.0 pM or less) is useful for an immuno-based methodfor determining BoNT/A activity described in Ester Fernandez-Salas, etal., Immuno-Based Botulinum Toxin Serotype A Activity Assays, U.S.patent application Ser. No. 12/403,531.

TABLE 3 Primary Screen for Clonal Cell Lines Susceptible to BoNT/AIntoxication BoNT/A Activity Clone EC₅₀ (U/mL) Signal_(MIN) (RLU)Signal_(MAX) (RLU) SiMa 10.8 ± 1.0 10,519 253,484 H1 10.2 ± 0.6 13,996276,982 H10 22.2 ± 2.0 5,379 296,168 1D4 22.4 ± 0.8 6,064 239,096 1E1114.2 ± 1.7 8,698 276,323 2D6 27.3 ± 1.8 3,347 179,454 2F5 15.8 ± 0.97,007 265,461 3D8 13.4 ± 0.6 11,658 266,279 YB8 19.7 ± 1.3 8,477 293,559

This initial primary screen using a dose-response curve with the 10different BoNT/A concentrations identified three concentrations thatwere useful for a shortened assay using 3 different BoNT/Aconcentrations, with one concentration being slightly higher than thelower asymptote (8 U/mL), one concentration near the EC₅₀ (25 U/mL), andone concentration representing about 80% of the upper asymptote (80U/mL). In these subsequent primary screens the remaining 29 clonal cellline isolates were tested as described above except that the cells weretreated with either 0 (untreated sample), 8 U/mL, 25 U/mL, or 80 U/mL ofa formulated BoNT/A pharmaceutical product. The clonal cell lines testedwere 1E3, 2B9, 2D2, 2E4, 3B8, 3D5, 3G10, 4B5, 4C8, 4D3, 5C10, 5F3, AC9,AF4, BB3, BB10, BE3, BF8, CC11, CD6, CE6, CG8, CG10, DC4, DD10, DE7,DF5, YB7, and YF5. In addition, the parental SiMa was used as positivecontrol.

These results show that clonal cell lines 2E4, 3B8, 3D5, 5F3, AF4, BB3,BB10, and DC4 all comprise cells that are more susceptible to BoNT/Athan the parental SiMa cell line (Table 4). In addition, these resultsindicate that clonal cell lines 1E3, 2B9, 3G10, 4B5, 4C8, AC9, BE3, BF8,CC11, CD6, CE6, CG8, CG10, DD10, DE7, and YF5 all comprise cells thatare of similar susceptibility to BoNT/A to that of the parental SiMacell line (Table 4). Lastly, these results reveal that clonal cell lines2D2, 4D3, 5C10, DF5, and YB7 all comprise cells that are lesssusceptible to BoNT/A than the parental SiMa cell line (Table 4).

TABLE 4 Primary Screen for Clonal Cell Lines Susceptible to BoNT/AIntoxication 8 U/mL BoNT/A 25 U/mL BoNT/A 80 U/mL BoNT/A Activity SignalFold Activity Signal Fold Activity Signal Fold Clone (RLU) Difference(RLU) Difference (RLU) Difference SiMa 75811/80562* — 138952/175685* —173979/225768* — 1E3 71040 0.9 128771 0.9 155107 0.9 2B9 66962 0.9135006 1.0 166871 1.0 2D2  60224* 0.8  136947* 0.8  199786* 0.9 2E4103680  1.4 217980 1.6 270463 1.6 3B8 123521  1.6 227367 1.6 249218 1.43D5 113532  1.5 201683 1.5 270578 1.6 3G10 83049 1.1 130930 0.9 1755401.0 4B5 74344 1.0 137604 1.0 177363 1.0 4C8 87047 1.1 144395 1.0 1851151.1 4D3 38063 0.5  80180 0.6 101724 0.6 5C10  16024* 0.2  49030 0.3 116396* 0.6 5F3  98263* 1.7  182902* 1.4  212275* 1.5 AC9  60016* 1.0 127030* 1.0  180384* 1.4 AF4 115460  1.5 179901 1.3 190867 1.1 BB374668 1.3 120966 1.4 170177 1.4 BB10 152748  2.0 232406 1.7 266555 1.5BE3 100849  1.0 199487 0.9 236415 1.0 BF8  55763* 1.0  111746* 1.0 153074* 1.3 CC11  54421* 1.0  123576* 1.0  155337* 1.2 CD6 74970 1.0151862 1.1 183377 1.1 CE6 70019 0.9 146292 1.1 208184 1.2 CG8  81790*1.0  159098* 1.0  189209* 1.3 CG10 69503 0.9 141079 1.0 185801 1.1 DC4112085  1.5 163319 1.2 169352 1.0 DD10 116325  1.5 205445 1.5 223473 1.3DE7  60994* 0.9  131237* 0.9  174487* 0.9 DF5  34,165* 0.5  104,696* 0.7 179,314* 0.9 YB7 50433 0.7 103707 0.7 143423 0.8 YF5 65848 0.9 1224870.9 146263 0.4 *Two separate screens were done. In the first, the signaldetected for the SiMa cell line was 75811 RLU and this value was used todetermine the fold difference for clonal cell lines tested in thisscreen (numbers with no asterisk). For the second screen, the signaldetected for the SiMa cell line was 80562 RLU and this value was used todetermine the fold difference for clonal cell lines tested in thisscreen (numbers with asterisk).

Example II Stability Analysis of Clonal Cell Lines

The following example illustrates how to characterize the stability ofcells from an established clonal cell line that are susceptible toBoNT/A intoxication or have neurotoxin uptake capacity.

During characterization of the SiMa cell line, it was determined thatthe parental SiMa cell line became unstable in terms of a significantloss of sensitivity to BoNT/A. For example, prior to passage 13, cellscomprising the parental SiMa cell line routinely exhibited an EC₅₀ forBoNT/A activity that was under 5.0 pM. However, after passages about 14to about 20 passages, cells comprising the parental SiMa cell exhibitedan EC₅₀ for BoNT/A activity that was routinely over 25.0 pM. One reasonfor this dramatic reduction in the EC₅₀ value may be because the SiMacell line is heterogeneous in nature since it comprises at least fivedifferent cell types. Over time, one of these cell types could overtakethe culture to the exclusion of the other four. If the overtaking celltype was less susceptible to BoNT/A intoxication, or not susceptible atall, then as this cell type became the dominant one in the cell line theEC₅₀ for BoNT/A activity in this overtaken SiMa cell line woulddecrease. Thus, because the established clonal cell lines were derivedfrom only one of these cell types, then it follows that the clonal cellline would exhibit greater stability in terms of maintaining an EC₅₀ forBoNT/A activity that was routinely under 5.0 pM.

To determine whether the cells from the established clonal cell linesshowed increased stability relative to the cells comprising the parentalSIMa cell line, the EC₅₀ for BoNT/A activity was determined for clonalcell lines BB10, H1, H10, and the parental SiMa cell line at differentpassage numbers using an immuno-based method for determining BoNT/Aactivity. The media from the differentiated cells was aspirated fromeach well and replaced with fresh media containing either 0 (untreatedsample), 0.98 U/mL, 1.9 U/mL, 3.91 U/mL, 7.81 U/mL, 15.6 U/mL, 31.3U/mL, 62.5 U/mL, 125 U/mL, 250 U/mL, or 500 U/mL of a formulated BoNT/Apharmaceutical product. After a 24 hrs treatment, the cells were washed,incubated for an additional two days in serum-free medium without toxinto allow for the cleavage of the SNAP-25 substrate, and harvested asdescribed above. The supernatant was transferred to a capture antibodycoated 96-well plate to perform the detection step.

Similarly, the preparation of the α-SNAP-25 capture antibody solution,α-SNAP-25 detection antibody solution, and the solid phase supportcomprising an α-SNAP-25 capture antibody were as described in Section 3.Lastly, detection of the presence of SNAP-25 cleavage product by ECLsandwich ELISA was performed, collected data was analyzed, and the EC₅₀calculated as described above in Section 2, except that PLA v. 2 wasused for the 4-parameter logistics fit.

The results from this stability experiments indicated that BB10, H1 andH10 cell lines all exhibited increase stability as compared to theparental SiMa cell line. For example, where cells from the parental SiMacell line exhibited an EC₅₀ for BoNT/A activity that was over 500 U/mL(or 25 pM) after passages about 14 to about 20 passages, cellscomprising the BB10, H1 and H10 cell lines all exhibited an EC₅₀ forBoNT/A activity that was below 10 U/mL (or 0.5 pM) after passage about30.

Example III Gene Expression Profile of Cell Lines Comprising CellsSusceptible to BoNT/A Intoxication

The following example illustrates how to characterize cells from anestablished clonal cell line that are susceptible to BoNT/A intoxicationor have neurotoxin uptake capacity by gene expression profiling.

To determine the changes in genome-wide gene transcription levels incells susceptible to BoNT/A intoxication, gene expression analysis wasperformed on cells from H1, BB10, 2D6 and the parental SiMa cell line,in both differentiated and undifferentiated states using GENECHIP® U133Plus 2 microarray technology (Affymetrix, Inc., Santa Clara, Calif.)according to the manufacturer's instructions, see, e.g., GENECHIP®Expression Analysis Technical Manual, 702232, Rev. 2 (2006) which ishereby incorporated by reference in its entirety.

To prepare differentiated cells for these cell lines, a suitable densityof cells from the H1, BB10, 2D6, and SiMa cell lines were seeded inseparate T175 tissue culture flasks containing 50 mL of a serum-freemedium comprising Enhanced Minimum Essential Medium, 10 mM HEPES, 1 mMsodium pyruvate, 0.1 mM Non-Essential Amino Acids, 1× B27 supplement,1×N2 supplement, and 60 μg/mL GT1b (302-0.1-M005, Alexis Biochemicals,San Diego, Calif.). These cells were incubated in a 37° C. incubatorunder 5% carbon dioxide until the cells differentiated, as assessed bystandard and routine morphological criteria, such as growth arrest andneurite extension, and attained 80% confluence (approximately 3 days).The cells were then washed with 50 mL of 1×PBS, trypsin-treated for 5min at room temperature, and then 15 mL of growth medium was added tothe flask to inactivate the trypsin. The cell suspension was transferredto a 50 mL tube and centrifuge at 200 RCF for 5 min at room temperatureto pellet the cells. The cell pellet was washed once in 50 mL of 1×PBS,centrifuge at 200 RCF for 5 min at room temperature to pellet the cells,and the supernatant was removed. The cell pellet was quick frozen on dryice and stored at −80° C. until needed.

To prepare undifferentiated cells for these cell lines, a suitabledensity of cells from the H1, BB10, 2D6, and SiMa cell lines were seededin separate T175 tissue culture flasks containing 50 mL of growth mediumcomprising RPMI 1640, 10% fetal bovine serum, 10 mM HEPES, 1 mM sodiumpyruvate, 0.1 mM Non-Essential Amino Acids, and 60 μg/mL GT1b. Thesecells were incubated in a 37° C. incubator under 5% carbon dioxide untilthe cells attained 80% confluence (approximately 3 days). The cells werethen processed as described above and the cell pellet was quick frozenon dry ice and stored at −80° C. until needed.

To prepare total RNA, cells for each cell line were lysed and total RNAisolated and purified using a RNA isolation kit (QIAGEN, Valencia,Calif.) according to the manufacturer's protocols. DNA was digestedusing an on-column DNase I procedure (QIAGEN, Valencia, Calif.). RNA wasquantified photospectrometrically at 260 nm and purity assessed by theA260A280 ratio using a spectrophotometer. RNA integrity was assessedusing a Bioanalyser 2100 (Agilent Technologies, Inc., Santa Clara,Calif.). Extracted total RNA aliquots were snap-frozen in liquidnitrogen and stored at −80° C.

To prepare cDNA probes for the gene microarray chips, about 1-15 μg wasreversed transcribed using a T7-Oligo(dT) promoter primer in the firststrand cDNA synthesis reaction. Following RNase H-mediated second strandcDNA synthesis, the double-stranded cDNA was purified and served as atemplate for subsequent in vitro transcription reaction. The in vitrotranscription reaction was carried out in the presence of T7 RNApolymerase and a biotinylated nucleotide analog/ribonucleotide mix forcomplementary RNA (cRNA) amplification and biotin labeling. Thebiotinylated cRNA targets were then cleaned up, fragmented, andhybridized to microchip expression arrays for 17 hours at 65° C.according to the manufacturer's instructions, see, e.g., GENECHIP®Expression Analysis Technical Manual, 702232, Rev. 2 (2006) which ishereby incorporated by reference in its entirety. To identifysubstantial treatment-related up- or down-regulated gene expressiondifferences compared to control cell lines, greater than 1.5-folddifferences (log₂ 0.58) were considered significant, and only thosegenes that displayed consistent changes in expression in the triplicateanalysis were considered (Tables 5-12).

Expression profiling for cell lines H1, BB10 and 2D6 analyzed in theundifferentiated state revealed that 1,323 genes were identified ashaving their expression levels increase by 1.5-fold (log₂ 0.58) or morein the H1 and BB10 cell lines as compared to the gene expression levelsobtained from the 2D6 cell line (Table 5). Of these, 686 genes exhibitedan increase in expression levels in the H1 and BB10 cell lines by 2-fold(log₂ 1.0) or more as compared to the expression level for these genesdetermined from the 2D6 cell line; and 231 genes exhibited an increasein expression levels in the H1 and BB10 cell lines by 4-fold (log₂ 2.0)or more as compared to the expression level for these genes determinedfrom the 2D6 cell line. With respect to decreased expression, 1,438genes were identified as having their expression levels decreased by1.5-fold (log₂ 0.58) or more in the H1 and BB10 cell lines as comparedto the gene expression levels obtained from the 2D6 cell line (Table 6).Of these, 668 genes exhibited a decrease in expression levels in the H1and BB10 cell lines by 2-fold (log₂ 1.0) or more as compared to theexpression level for these genes determined from the 2D6 cell line; and107 genes exhibited a decrease in expression levels in the H1 and BB10cell lines by 4-fold (log₂ 2.0) or more as compared to the expressionlevel for these genes determined from the 2D6 cell line.

Expression profiling for cell lines H1, BB10 and 2D6 analyzed in thedifferentiated state revealed that 2,556 genes were identified as havingtheir expression levels increased by 1.5-fold (log₂ 0.58) or more in theH1 and BB10 cell lines as compared to the gene expression levelsobtained from the 2D6 cell line (Table 7). Of these, 1,634 genesexhibited an increase in expression levels in the H1 and BB10 cell linesby 2-fold (log₂ 1.0) or more as compared to the expression level forthese genes determined from the 2D6 cell line; and 696 genes exhibitedan increase in expression levels in the H1 and BB10 cell lines by 4-fold(log₂ 2.0) or more as compared to the expression level for these genesdetermined from the 2D6 cell line. With respect to decreased expression,2,087 genes were identified as having their expression levels decreasedby 1.5-fold (log₂ 0.58) or more in the H1 and BB10 cell lines ascompared to the gene expression levels obtained from the 2D6 cell line(Table 8). Of these, 1,215 genes exhibited a decrease in expressionlevels in the H1 and BB10 cell lines by 2-fold (log₂ 1.0) or more ascompared to the expression level for these genes determined from the 2D6cell line; and 280 genes exhibited a decrease in expression levels inthe H1 and BB10 cell lines by 4-fold (log₂ 2.0) or more as compared tothe expression level for these genes determined from the 2D6 cell line.

Expression profiling for cell lines H1, BB10 and SiMa analyzed in theundifferentiated state revealed that 1,232 genes were identified ashaving their expression levels increase by 1.5-fold (log₂ 0.58) or morein the H1 and BB10 cell lines as compared to the gene expression levelsobtained from the SiMa parental cell line (Table 9). Of these, 764 genesexhibited an increase in expression levels in the H1 and BB10 cell linesby 2-fold (log₂ 1.0) or more as compared to the expression level forthese genes determined from the SiMa parental cell line; and 228 genesexhibited an increase in expression levels in H1 and BB10 cell lines by4-fold (log₂ 2.0) or more as compared to the expression level for thesegenes determined from the SiMa parental cell line. With respect todecreased expression, 1,265 genes were identified as having theirexpression levels decreased by 1.5-fold (log₂ 0.58) or more in the H1and BB10 cell lines as compared to the gene expression levels obtainedfrom the SiMa parental cell line (Table 10). Of these, 648 genesexhibited a decrease in expression levels in the H1 and BB10 cell linesby 2-fold (log₂ 1.0) or more as compared to the expression level forthese genes determined from the SiMa parental cell line; and 189 genesexhibited a decrease in expression levels in H1 and BB10 cell lines by4-fold (log₂ 2.0) or more as compared to the expression level for thesegenes determined from the SiMa parental cell line.

Expression profiling for cell lines H1, BB10 and SiMa analyzed in thedifferentiated state revealed that 756 genes were identified as havingtheir expression levels increase by 1.5-fold (log₂ 0.58) or more in theH1 and BB10 cell lines as compared to the gene expression levelsobtained from the SiMa parental cell line (Table 11). Of these, 472genes exhibited an increase in expression levels in the H1 and BB10 celllines by 2-fold (log₂ 1.0) or more as compared to the expression levelfor these genes determined from the SiMa parental cell line; and 150genes exhibited an increase in expression levels in H1 and BB10 celllines by 4-fold (log₂ 2.0) or more as compared to the expression levelfor these genes determined from the SiMa parental cell line. Withrespect to decreased expression, 912 genes were identified as havingtheir expression levels decreased by 1.5-fold (log₂ 0.58) or more in theH1 and BB10 cell lines as compared to the gene expression levelsobtained from the SiMa parental cell line (Table 12). Of these, 411genes exhibited a decrease in expression levels in the H1 and BB10 celllines by 2-fold (log₂ 1.0) or more as compared to the expression levelfor these genes determined from the SiMa parental cell line; and 108genes exhibited a decrease in expression levels in H1 and BB10 celllines by 4-fold (log₂ 2.0) or more as compared to the expression levelfor these genes determined from the SiMa parental cell line.

Example IV Pathway Analysis of Gene Expression Profiles from Clonal CellLines

The following example illustrates how to characterize gene expressionprofiles from clonal cells from an established clonal cell line that aresusceptible to BoNT/A intoxication or have neurotoxin uptake capacity inorder to identify biological networks or pathways associated with BoNT/Aintoxication.

An Ingenuity Pathway Analysis (IPA) Core Analysis (Ingenuity SystemsInc., Redwood City, Calif.) was performed to characterize the RNAexpression phenotype of two independently derived BoNT/A very sensitivesingle clone cell lines, BB10 and H1, compared to a related but muchless BoNT/A sensitive single clone cell line, 2D6. The analysis was doneto identify the specific gene expression phenotype of a BoNT/A sensitivecell line. The result can also be used to predict and potentiallyidentify proteins that function to enhance BoNT/A uptake.

An Ingenuity Pathway Analysis (IPA) Core Analysis was performed thatidentified biologically relevant networks to BoNT/A intoxication basedon connectivity among the genes from an IPA library and the genesexhibiting significant changes as determined from the expressionprofiling discussed above. For this analysis, all genes that exhibitedan increased or decrease in expression levels in the H1 and BB10 celllines by 4-fold (log₂ 2.0) or more as compared to the expression levelfor these genes determined from the 2D6 cell line were used, inconjunction with, selected genes with known or suspected relevance toBoNT/A intoxication, hereafter referred to as the Profile genes. Thesignificance of the association to a given network was measured andranked by the ratio of the number of Profile genes that mapped to thespecific network divided by the total number of genes that map to thatnetwork. Fisher's exact test was used to calculate p-scores, whichrepresent the association between the Profile genes and the network, andbased on the p-scores the genes from the Profile genes were used to rankthe networks. The p-scores were derived from p-values. If there are ngenes in the network and f of them are Profile genes, then the p-valueis the probability of finding f or more Profile genes in a set of ngenes randomly selected from the Global Molecular Network. Sincesignificant p-values are quite low (e.g., 1×10⁻⁸), a p-score was definedas the component of the p-value; p-score=−log 10 (p-value). Theidentified networks were overlaid with differentially expressed geneswhich had more than 1.5-fold differential expression and a p-value 0.001either before or after differentiation.

Using IPA Core Analysis the differentially expressed Profile genes wereconnected to 26 Networks. Nineteen of these included 10 or more Profilegenes (Table 13). Among these nineteen networks three overall networksgroups, named A, B and C were identified (Table 13). Within the networksin these three groups there were overlapping genes and the expressionfor more than three genes was increased after differentiation. Group Agenes include the FGFR2 receptor which has been shown to function in theBoNT/A intoxication process as a receptor for BoNT/A and are as follows:genes exhibiting a 1.5-fold or more decrease in expression levels in theH1 and BB10 cell lines as compared to their expression in cells from the2D6 cell line, ANXA2, AQP1, ARHGAP9, CDH10, CDKN2A, CHPT1, CNTN2, ERAP1,and RGS11; genes exhibiting a 1.5-fold or more increase in expressionlevels in the H1 and BB10 cell lines as compared to their expression incells from the 2D6 cell line, ADAMTS9, ATAD2, C11ORF82, CDC45L, CNTN1,CNTN4, Cyclin A, Cyclin E, E2F1, E2F2, E2F7, ELOVL7, EME1, FGFR, FGFR2,KIAA1524, MELK, MYBL1, MYBL2, NDC80, NDN, ORC1L, PLS3, PRIMAL, RAD54L,RBL1, RBPMS, RRM2, S1PR3, SCLY, SLCIA3, SPC24, SPC25, ST8SIA4, TFDP1,TFP12, TK1, TMEM35, TTK, TWIST1, TYMS, TYK, and ZWINT.

Group B genes include the EGFR, a receptor which is regulated by NGF,which has been shown to increase BoNT/A uptake in cell cultureexperiments and are as follows: genes exhibiting a 1.5-fold or moredecrease in expression levels in the H1 and BB10 cell lines as comparedto their expression in cells from the 2D6 cell line, ABCC8, AELIM3,CAP2, IL17B, MEF2A, NEEBL, PHC, S100A6, SLC1A6, SMAD1, SMAD5, SMAD8,SYT13, and SYTL1; genes exhibiting a 1.5-fold or more increase inexpression levels in the H1 and BB10 cell lines as compared to theirexpression in cells from the 2D6 cell line, AURKB, BIRC5, BRCA1, BRCA2,BRIP1, BUB1B, CD9, DLGAP3, DYNLT3, ENC1, FBLN1, FOXM1, Gβγ, GNAI1,GNG11, GNG12, GPSM2, GUCY1B3, HGF, ITGA6, JNK, KCNJ5, KIF18A, KITLG,MMD, MSN, MYRIP, NEK2, NR3Cl, NXPH1, OSBPL3, PKMYT1, PTPRM, RAD51,RAD51AP1, SLC7A2, SLC43A3, SMC6, SNAI2, SNCAIP, SSH2, STK17A, SYNPO2,TOP2A, TPTE, TRAF4, TSPAN, TSPAN4, UBE3, UBE3B, and VAV3.

Group C genes include a number of microtubule motor proteins of thekinesin family that may function in the intracellular trafficking ofBoNT/A and are as follows: genes exhibiting a 1.5-fold or more decreasein expression levels in the H1 and BB10 cell lines as compared to theirexpression in cells from the 2D6 cell line, CSTB, GPCR, GRIM5, KISSR,SCN2A, SLC1A2, and THBS2; genes exhibiting a 1.5-fold or more increasein expression levels in the H1 and BB10 cell lines as compared to theirexpression in cells from the 2D6 cell line, ALCAM, AURKA, CHEK, CIT,CSRP2, E2F, ECT2, EFNB2, ERK, ESPL1, GNAI, GPR161, HMMR, KIF4A, KIF14,KIF15, KIF22, KIF23, KIFC1, LPAR1, MK167, OIP5, PHLPP, PP1/PP2A,PPP1R3C, PRC1, PTTG1, RACGAP, RB, RGS5, SDC2, and TPX2.

The remaining networks identified did not share common genes between anyother network. For Network 3, genes exhibiting a 1.5-fold or moredecrease in expression levels in the H1 and BB10 cell lines as comparedto their expression in cells from the 2D6 cell line, ADARB1, ADM, PTPPH,and SLCO1A2; genes exhibiting a 1.5-fold or more increase in expressionlevels in the H1 and BB10 cell lines as compared to their expression incells from the 2D6 cell line, ASCL1, HES6, MAPK, NMU, PEG3, PTPRK,PRLHR, PTPRK, SGOL2, SPARC, and ZNF217. For Network 6, genes exhibitinga 1.5-fold or more decrease in expression levels in the H1 and BB10 celllines as compared to their expression in cells from the 2D6 cell line,CNGA3, HIST1H3E and PTGS1; genes exhibiting a 1.5-fold or more increasein expression levels in the H1 and BB10 cell lines as compared to theirexpression in cells from the 2D6 cell line, ASF1B, BASP1, CHAF1A, NCAPH,PBK, PRAME, SMC2, UHRF1, and VRK1. For Network 9, genes exhibiting a1.5-fold or more decrease in expression levels in the H1 and BB10 celllines as compared to their expression in cells from the 2D6 cell line,HIST1 H2BD and OSCAR; genes exhibiting a 1.5-fold or more increase inexpression levels in the H1 and BB10 cell lines as compared to theirexpression in cells from the 2D6 cell line, C14ORF106, CEP72, KIF20A,PCNA, PEX13, PFCS, POLQ, SPAGS, SYTL4, TROAP, and WDR51A. For Network12, genes exhibiting a 1.5-fold or more increase in expression levels inthe H1 and BB10 cell lines as compared to their expression in cells fromthe 2D6 cell line, ANLN, ARHGAP24, ASPM, BUB1, CCDC99, CEP55, CKAP2,DRAM, E2F8, PLXNA2, SLC16A10, UBE2C, UBE2S, and WDHD1. For Network 13,genes exhibiting a 1.5-fold or more decrease in expression levels in theH1 and BB10 cell lines as compared to their expression in cells from the2D6 cell line, CALY; genes exhibiting a 1.5-fold or more increase inexpression levels in the H1 and BB10 cell lines as compared to theirexpression in cells from the 2D6 cell line, ARHGEF3, CTSL2, DIAPH3,FBP1, KIF2C, KIF11, PFKFB3, and PLK4. For Network 14, genes exhibiting a1.5-fold or more decrease in expression levels in the H1 and BB10 celllines as compared to their expression in cells from the 2D6 cell line,COL5A1 and MICAL2; genes exhibiting a 1.5-fold or more increase inexpression levels in the H1 and BB10 cell lines as compared to theirexpression in cells from the 2D6 cell line, FNDC5, HSPC159, MAB21L2,SLITRK5, SYN2, and ZNF367. For Network 15, genes exhibiting a 1.5-foldor more decrease in expression levels in the H1 and BB10 cell lines ascompared to their expression in cells from the 2D6 cell line, OLFML2Aand SIGIRR; genes exhibiting a 1.5-fold or more increase in expressionlevels in the H1 and BB10 cell lines as compared to their expression incells from the 2D6 cell line, EXO1, KCTD12, MYO6, PHEBL1, SHCBP1, TPBG,and TUBB6. For Network 17, genes exhibiting a 1.5-fold or more decreasein expression levels in the H1 and BB10 cell lines as compared to theirexpression in cells from the 2D6 cell line, HPCAL1 and LPAR5; genesexhibiting a 1.5-fold or more increase in expression levels in the H1and BB10 cell lines as compared to their expression in cells from the2D6 cell line, BTG3, GABRA5, TR1P10, and ZNF521. For Network 19, genesexhibiting a 1.5-fold or more decrease in expression levels in the H1and BB10 cell lines as compared to their expression in cells from the2D6 cell line, HTR1E and SORCS1; genes exhibiting a 1.5-fold or moreincrease in expression levels in the H1 and BB10 cell lines as comparedto their expression in cells from the 2D6 cell line, BNC2, DPYD,EMILIN2, PPIL5, and TACC3.

TABLE 13 IPA Core Analysis of Profile Genes Profile ExpressionExpression Differentiated Proteins in Gene Undifferentiated State StateGroup Network Network pvalue Number Decreased Increased DecreasedIncreased A 1 ADAMTS9, 51 30 CDKN2A ADAMTS9, CDKN2A, ADAMTS9, ATAD2,CNTN1, CNTN2, ATAD2, CDC45L, HDAC, NDN, CDC45L, CDKN2A, PLS3, CNTN1,CNTN1, S1PR3, TFPI2 Cyclin A, CNTN2, Cyclin Cyclin E, A, Cyclin E, E2F1,E2F2, DIRAS3, E2F1, E2F7, E2F2, E2F7, FGFR2, FGFR2, HDAC, MYBL1, MYBL1,MYBL2, MYBL2, NDC80, NDC80, NDN, NDN, NFkB, ORC1L, ORC1L, PLS3, PLS3,RAD54L, RAD54L, RBL1, RRM2, RBL1, S1PR3, RRM2, SPC24, S1PR3, SPC25,SPC24, TFDP1, TFPI2, SPC25, TK1, TFDP1, TNFRSF10D, TFPI2, TK1, TTK,TYMS, TTK, TYMS, TYK, ZWINT TYK, ZWINT C 2 ALCAM, ANK2, 46 28 ANK2,ALCAM, KISSR, ALCAM, ANK3, AURKA, ANK3, DOK5, ERK, SCN2A AURKA, DOK5,E2F, PHKA2, GFRA2, E2F, ERK, ERK, ESPL1, PPP2R2C, PP1/PP2A, ESPL1,GDF15, SCN2A PP2A, HMMR, GFRA2, PPP1R3C, KIF15, HMMR, ITGA9, PPP2R2BKIF22, KIF15, KIF22, MKI67, OIP5, KISS1R, PHLPP, KPNA, KPNB, PP1/PP2A,LGI1, MAFB, PPP1R3C, MAP2K1, PTTG1, RB, MAP2K2, TPX2 MKI67, NRG3, OIP5,PHKA2, PHLPP, POSTN, PP1/PP2A, PP2A, PPP1R3C, PPP2R2B, PPP2R2C, PTTG1,RAF1, RB, SCN2A, TPX2 — 3 ADARB1, 46 28 ADARB1, ASCL1, ADARB1, ASCL1,ADM, AKAP12, GRIP2, DLL1, FRZB, ADM, HES6, ALP, ASCL1, SLCO1A2 MAPK,PTPPH, MAPK, NMU, BHLHB2, CRH, PEG3, PLAT, SLCO1A2 PEG3, DLL1, FRZB,PRLHR, PRLHR, FSH, GRIA2, SMAD6, PTPRK, GRIP2, hCG, SPARC, SGOL2, HES6,ZNF217 SPARC, HTATIP2, ZNF217 IL13RA2, MAPK, NMU, PDGF BB, PDLIM3, PEG3,PLAT, PPFIA4, PRLHR, PTPRH, PTPRK, SGOL2, SLCO1A2, SMAD6, SPARC, STC1,TEAD4, TGFB, VEGF, ZNF217 C 4 AKT, ACTN1, 43 27 CSTB, GNAI, CSTB, CIT,CSRP2, ACTN2, GPCR, GPR161, GPCR, ECT2, ACTN3, ATF5, GRIM5, LPAR1,GRIM5, EFNB2, ATM/ATR, SLC1A2 RGS5 SLC1A2, CHEK, GNAI, BDKRB2, THBS2GPR161, CHEK1, KIF4A, CHEK2, CIT, KIF14, CSRP2, CSTB, KIF23, CTSH, ECT2,KIFC1, EFNB2, LPAR1, ELMO1, GNAI, PRC1, GABBR2, RACGAP, GPCR, RGS5,GPR161, SDC2, GRM5, ICAM2, KIF4A, KIF14, KIF23, KIFC1, LPAR1, MMP, PDGFAPDGFB, PRC1, RAC, RAC2, RACGAP1, RGS5, SDC2, SLC1A2, THBS2 B 5 ABLIM3,F- 38 25 SYT13, CD9, CXCR4, AELIM3, CD9, ENC1, ACTF, G-ACT, S100A6 ENC1,Gβγ, S100A6, Gβγ, GNAI1, CD9, CXCR4, GNAI1, SYT13, GNG11, DTNA, ENC1,GNG11, SYTL1 GNG12, FNBP1, Gβγ, GNG12, GPSM2, GNAI1, ITGA6, JNK, ITGA6,JNK, GNG11, MSN, MYRIP, KCNJ4, GNG12, STK17A MSN, GPSM2, ITG, MYRIP,ITA5, ITGA6, NXPH1, JNK, KCNJ4, STK17A, MLC, MSN, TRAF4, MYRIP, NOX,TSPAN, NET1, NRXN1, TSPAN4, NXPH1, PI3K, VAV3 RHO, S100A6, STK17A,SYT13, SYTL1, TRAF4, TSPAN, TSPAN3, TSPAN4, VAV3 — 6 ADCY, ADCY1, 36 24CAMK2B BASP1, CNGA3, ASF1B, AP1, ASF1B, HRH3 HIST1H3E, BASP1, ATP2B4,PTGS1 CHAF1A, BASP1, NCAPH, BCL11B, CAM, PBK, CAMK2B, PRAME, CHAF1A,SMC2, CNGA3, UHRF1, CYP2E1, VRK1 DACH1, HIST1H3E, HISH3, HRH3, HSP90,IL1, IL12, INS, INFA, ISG20, KCNN2, LDL, MMP14, NCAPH, PBK, PDGF, PIAS1,PRAME, PTGS1, SEC31A, SMC2, UHRF1, VRK1 B 7 BARD1, 32 24 CAP2, NR3C1,CAP2, BRCA1, BRCA1, CNA, SMC6, MEF2A, BRCA2, BRCA2, PHLDA2, SNCAIP,PKC(s), BRIP1, BRIP1, PKC(s) TCF7L2 SLC1A6 BUB1B, BUB1B, CNA, DLGAP3,CAP2, CK2, GUCY1B3, DLGAP5, FAT, NEK2, GUCY1B3, KITLG, HSP70, KITLG,NR3C1, MEF2A, NEK2, RAD51, NFAT, NR3C1, RAD51AP1, NF1, PHLDA2, SMC6,PKC(s), SNAI2, RAD51, SNCAIP, RAD51AP1, UBE3, TOP2, UBE3B SAMD4A,SLC1A6, SMC6, SNAI2, SNCAIP, STAT5a, STAT5b, TCF7L2, UBE3, UBE3B, UBN,XPO1 B 8 CBP, AURKB, 30 21 KCNMA1, ERK1, ERK2, NDRG1, AURKB, BIRC5,NDRG1, MAOA, P38 SMAD1, BIRC5, CACNA1D, RAS MAPK, SMAD5, ERK1, ERK2,CBP/p300, RGMA SMAD8 FBLN1, CD3, CREB, FOXM1, CREB5, DBH, HGF, MAOA,EGR1, ERK1, P38 MAPK, ERK2, FBLN1, PKMYT1, FBN1, FOXM1, SYNPO2, HGF,INCENP, TOP2A KCNMA1, LGALS1, MAOA, MEK, NDRG1, NID1, P38 MAPK, PKA,PKMYT1, PRKAC, PRKACB, RAF, RAP1, RAS, RGMA, RSK, SMAD1, SMAD5, SMAD8,SYNPO2, TOP2A — 9 C14ORF106, 19 15 — PCGF5 HIST1H2BD, C14ORF106, CDKN1A,OSCAR CEP72, CEP72, DHX8, KIF20A, FKBPL, PCNA, HIST1H2BD, PEX13, HNF4A,PFC5, KIF20A, POLQ, KRT18, SPAG5, MTHFS, SYTL4, OSCAR, TROAP, PCGF5,PCNA, WDR51A PELO, PEX13, PFC5, POLD4, POLK, POLL, POLM, POLQ, POLS,R3HDM1, RASL12, RFC5, SLC33A1, SPAG5, STAT4, SULT1A1, SULT1C2, SYTL4,TCEAL3, TROAP, VEZT, WDR51A, ZBTB16 A 10 ARHGAP9, 18 15 CHPT1 CNTN4,ARHGAP9, CNTN4, CDH10, CDS1, ELOVL7, CDH10 ELOVL7, CDS2, CHPT1, HAND1,EME1, CNTN4, ODS2, SOX2, FGFR, ELOVL7, TWIST1 KIAA1524, EME1, FGFR,PRIMA1, GAS6, HAND1, RBPMS, IFI202B, ISL1, ST8SIA4, KIAA1524, TWIST1KRT74, MIRN31, MUS81, MYF6, MYOD1, NFYB, OTX1, PRIMA1, QKI, RBM9, RBPMS,RORA, SHOX2, SLC39A8, SOX2, SOX15, ST8SIA4, STAT3, TWIST1, VPS39, ZFHX3B 11 ALDH3A2, 18 15 PHLDA2 BAMBI, — DYNLT3, B3GALT2, CUGBP2, KIF18A,BAMBI, CNN2, DYNLT3, PTPRM, CTNNAL1, SLC7A2, SLC7A2, CTNNB1, SLC43A3,SLC43A3, CTSC, STK17A, SSH2, CUGBP2, TPTE STK17A, DYNLT3, IL13, TPTEKIF18A, LIN7C, MFI2, MPP5, MRC2, NDST1, PDGF- CC, PHLDA2, PLAU, PPM1J,PPP1CA, PTPN14, PTPRM, SLC16A3, SLC26A2, SLC43A3, SLC7A2, SSH2, STK17A,TAX1BP3, TCF7L1, TGFB1, TPTE, TSPAN8 — 12 AHR, AHRR, 17 15 HS3ST1PDZRN3, — ANLN, ANLN, APH1B, PVRL3 ARHGAP24, ARHGAP24, ASPM, ASPM, BUB1,BUB1, CCDC99, CCDC99, CEP55, CEP55, CKAP2, CKAP2, DRAM, E2F8, DRAM,HS3ST1, E2F8, JPH1, PLXNA2, MIRN124-1, SLC16A10, NMT2, UBE2C, PDZRN3,UBE2S, PLXNA2, WDHD1 PMM1, PPP1R13L, PVRL3, RFFL, S100A2, SLC16A10,TP53, UBE2A, UBE2C, UBE2D2, UBE2S, UBE2V1, UBE2V2, UBL3, VPS37C, WDHD1 —13 ARHGEF3, 17 14 CALY ARHGEF3, CALY ARHGEF3, CALY, CSH2, GSK3B, CTSL2,CTSL2, RMP22 DIAPH3, DIAPH3, FBP1, ECEL1, FBP1, KIF2C, FFAR2, KIF11,FRAT2, PFKFB3, GSK3B, PLK4 HSD17B4, IL6, IL19, KIF11, KIF2C, KNG1,LARGE, LEFTY2, LGR5, LIF, LPAR3, MPZ, MSLN, ORM2, PFKFB3, PLK4, PMP22,PRPH, PRSS8, PTGER3, SCN7A, SCNN1B, SLC4A11, TAL1 — 14 AGT, 15 13 CPNE3CADPS, COL5A1, FNDC5, AGTRAP, Beta FNDC5, MICAL2 HSPC159, ARK, CADPS,MAB21L2, MAB21L2, CD160, SLITRK5, SLITRK5, COL5A1, ST8SIA2 SYN2, CPNE8,ZNF367 FNDC5, HSPC159, INA, ITGA7, LIMA1, LPP, MAB21L2, MAPK1, MERTK,MICAL2, MIRN294, MIRN185, MIRN352, NTN1, PLCG1, PLCG2, PTGFR, RASGEF1B,RBM4B, RSU1, SKAP1, SLITRK5, SPRED1, ST8SIA2, SYN2, TGFB3, TNS1, ZNF367— 15 ACT1, BAG5, 15 13 — KCTD12, OLFML2A, EXO1, CPVL, EXO1, TPBG SIGIRRKCTD12, GIPC1, GJC1, MYO6, GPR37, PHEBL1, HSPA5, SHCBP1, HSPA1A, TPBG,HSPBP1, TUBB6 IkBKB, KCTD12, MAP3K3, MAP3K14, MEKK3/NIK, MIRN30B, MYO6,OLFML2A, PELI3, RHEBL1, RNF126, RPL10A, RPN1, SEC16A, SHC1, SHCBP1,SIGIRR, TJP3, TPBG, TRAF6, TRAF2-TRAF5, TRAF2-TRAF5, TRAF6, TUBB6,TUBB2B B 16 ABCC8, 15 13 IL17B DACT1, ABCC8, MMD, BACE2, PCDH8 IL17B,OSBPL3, CLASP1, NEEBL SYNPO2 COLEC12, CXCR7, DACT1, ENTPD5, FGD6, FMO1,GSTT1, IL17B, KRT34, KYNU, LAMP3, LMCD1, MIRN101B, MMD, NEBL, NFRKB,OSBPL3, PCDH8, PEMT, RASAL2, SAMD4A, ST3GAL3, SYNGR3, SYNPO2, TNF,TNNC1, TXN2, YWHAG, ZNF267, ZYX — 17 ARPP-19, 14 13 HPCAL1 PPAP2BHPCAL1, BTG3, BTG3, LPAR5 GABRA5, CBFA2T2, TRIP10, CBLN1, ZNF521 CNGA2,EBF1, EGF, EGFR, GABA, GABRA5, GABRB3, GABRD, GABRR1, HDAC9, HPCAL1,HTT, IDS, INSL3, LPAR5, OPRL1, PCDH9, PDE10A, PDE11A, PDE1A, PDE1B,PDE2A, PDE4C, PDE6H, PPAP2B, PPP1R1B, ROBO2, SH3D2C1, TRIP10, ZNF521 A18 ANXA2, AQP1, 14 12 AQP1, MINA, ANXA2, C11ORF82, C11ORF82, CDKN2APDE5A CDKN2A, MELK, MINA, CCNI, ERAP1, PDE5A, CDKN2A, RGS11 SCLY,DEFB104A, SLC1A3, DEFB4, TMEM35 EGLN3, ERAP1, FAM129A, GNAO1, IFNG,IL1B, IL1F7, INCA, LOC729687, MELK, MINA, MIRN210, MPA2L, MYC, NNAT,PDE5A, RGS11, RNASE7, RT1- B, SCLY, SCUBE1, SLC11A1, SLC14A1, SLC1A3,ST18, TSH, TMEM35, TREM3 — 19 BIRC8, BNC2, 10 10 — EMILIN2, HTR1E, BNC2,CASP5, PLXNA4 SORCS1 DPYD, CASP10, EMILIN2, CASP14, PPIL5, CASP, CBLN2,TACC3 DEDD2, DNASE1L1, DPYD, DSG3, EIF4E, EMILIN2, HTR1D, HTR1E, HTR1F,HTR3A, IFT57, KHDC1, MIRN20A, MTCH1, NGFR, PLXNA4, PPIL5, RB1,RP11-257K9.7, SEMA6D, SERPINA3K, SORCS1, STK25, SYT5, TACC3, TERT — 20EGFL7, GFI1B 2 1 — — — — — 21 AGRN, 2 1 — — — — PRSS12 — 22 KCNB1, 2 1 —— — — KCNG3 — 23 NUP37, 2 1 — — — — NUP43 — 24 HS6ST1, 2 1 — — — —HS6ST2 — 25 FUCA1, 2 1 — — — — FUCA2 — 26 IQGAP, 2 1 — — — — IQGAP3,MIRN339

Example V Pathway Analysis of Gene Expression Profiles from Clonal CellLines

The following example illustrates how to characterize gene expressionprofiles from clonal cells from an established clonal cell line that aresusceptible to BoNT/A intoxication or have neurotoxin uptake capacity inorder to identify biological networks or pathways associated with BoNT/Aintoxication.

Computer analysis was performed to characterize the RNA expressionphenotype of two independently derived single clone cell lines, BB10 andH1, very sensitive to BoNT/A activity and a single clone cell line, 2D6,that was less sensitive BoNT/A activity. The results are useful inidentifying clonal cell lines selected from the SiMa parental cell linethat are useful to practice the methods disclosed in the presentspecification.

To identify the specific gene expression phenotype associated with aBoNT/A sensitive cell line, the data was analyzed using JMP Genomic (SASInstitute Inc., Cary, N.C.). To perform a JMP Genomics analysis, the RNAexpression data was imported to JMP Genomics by creating two SAS files,designated “data” and “Experiment design.” A basic expression workflowwas performed, including a variance analysis and one-way analysis ofvariance (one-way ANOVA) to identify differences in mean expressionvalues. The variance analysis was used to identify differences betweenthe cell lines and the differentiation state of each cell line(differentiated or non-differentiated). The expression values of allfour cell lines were compared pair wise, independent of differentiationand genes that were 4-fold over-expressed in the H1 cell line ascompared to the 2D6 cell line, in the BB10 cell line as compared to the2D6 cell line, and in SiMa parental cell line (PA) compared to the 2D6cell line. The variance analysis treats all factors in the study asrandom effects, to find out what their contribution is to the proportionof variance explained (total variance=100%). About half of the variance,46.9% was assigned to differences among the different cell types, while26% was assigned to differences before and after differentiation. Only4.6% of the variance was assigned to differences among differentiated ornon-differentiated cells types, meaning that the genes that weredifferentially expressed after differentiation were similar for all celltypes examined, suggesting that the difference in BoNT/A sensitivityamong the cell lines is independent of differentiation state. As such,the focus was placed on genes that are different among the differentcells types irrespective of differentiation.

Based on the results from the variance analysis, the expression valuesof all cell lines were compared pair wise, independent ofdifferentiation (FIG. 2). Only gene probes that were 4-foldover-expressed in the H1 cell line as compared to the 2D6 cell line, andin the BB10 cell line as compared to the 2D6 cell line are shown. Thegene probes were plotted as expression value (Log₂) on the x-axis andp-value (−log₁₀(p-value)) on the y-axis. The red dashed line marks the95% confidence interval. The genes probes that were ≧4-foldover-expressed in the H1 cell line as compared to the 2D6 cell line(FIG. 2A), and the BB10 cell line as compared to the 2D6 cell line (FIG.2B), were also to a large extend among the genes that wereover-expressed in the BB10 cell line as compared to the SiMa parentalcell line (PA, FIG. 2C), in the H1 cell line as compared to the SiMaparental cell line (FIG. 2D), and in the SiMa parental cell line ascompared to the 2D6 cell line (FIG. 2E). These data suggest that BoNT/Asensitivity was related to a gradual increase in expression of thesegenes. Based on this finding, the number of gene probes was furthernarrowed down to 119 by including only gene probes that were alsoover-expressed in the SiMa parental cell line compared to the 2D6 cellline, rationalizing that gene probes that were differential expressedacross one more cell line would be even more likely to be important forBoNT/A sensitivity. As such, the JMP Genomic analysis resulted in theidentification of 119 gene probes, designated the JMP probe set.

The JMP probe set was exported to Ingenuity Pathway Analysis (IPA) CoreAnalysis (Ingenuity Systems Inc., Redwood City, Calif.) to furthercharacterize the JMP probe set. Using this analysis, biologicallyrelevant networks to BoNT/A intoxication were identified based onconnectivity among the genes from an IPA library and the gene probescontained in the JMP probe set. The significance of the association to agiven network was measured and ranked by the ratio of the number of geneprobes from the JMP probe set that mapped to the specific networkdivided by the total number of genes that map to that network. Fisher'sexact test was used to calculate p-scores, and based on the p-scores thegenes from the JMP probe set were used to rank the networks. Thep-scores were derived from p-values. If there are n genes in the networkand f of them are gene probes from the JMP probe set, then the p-valueis the probability of finding f or more gene probes from the JMP probeset in a set of n genes randomly selected from the Global MolecularNetwork. Since significant p-values are quite low (e.g., 1×10⁻⁸), ap-score was defined as the component of the p-value; p-score=−log 10(p-value). The identified networks were overlaid with differentiallyexpressed genes which had more than 1.5-fold differential expression anda p-value ≦0.001 either before or after differentiation. Using IPA CoreAnalysis, 111 of the 119 gene probes from the JMP probe set were mappedto networks. The eight unmapped probes most likely represent either aprobe with an outdated nomenclature or one representing a gene without aknown gene product. The 111 gene probes corresponded to 79 genes thatwere greater than 4.0-fold differentially expressed in the H1 and BB10cell lines when compared to the 2D6 cell line and that were also greaterthan 4.0-fold differentially expressed in the SiMa parental cell lineswhen compared to the 2D6 cell line, when the p-value cut off was set to0.05 (Table 14).

TABLE 14 Genes Identified using IPA Core Analysis Gene Log Gene NameSymbol Probe ID. No. Location Type Ratio Achaete-scute complex ASCL1209985_s_at Nucleus Transcription 4.469 homolog 1 regulatorAchaete-scute complex ASCL1 209988_s_at Nucleus Transcription 2.774homolog 1 regulator Achaete-scute complex ASCL1 213768_s_at NucleusTranscription 2.955 homolog 1 regulator Acyl-CoA thioesterase 9 ACOT9221641_s_at Cytoplasm Enzyme 2.251 ADAM metallopeptidase with ADAMTS9226814_at Extracellular Peptidase 4.034 thrombospondin type 1 motif, 9Space Ankyrin 2, neuronal ANK2 216195_at Plasma Other −2.673 MembraneAnkyrin 2, neuronal ANK2 232606_at Plasma Other −2.621 Membrane Bloodvessel epicardial BVES 228783_at Plasma Other 3.174 substance MembraneBrain abundant, membrane BASP1 202391_at Plasma Other 4.549 attachedsignal protein 1 Membrane BRCA1 associated RING BARD1 205345_at NucleusTranscription 3.44 domain 1 regulator Calsyntenin 2 CLSTN2 219414_atPlasma Transporter 2.024 Membrane cAMP responsive element CREB5232555_at Nucleus Transcription −2.67 binding protein 5 Cathepsin L2CTSL2 210074_at Cytoplasm Peptidase 2.119 CD9 molecule CD9 201005_atPlasma Other 2.887 Membrane Cell division cycle associated CDCA7L225081_s_at Nucleus Other 3.737 7-like Centromere protein L CENPL1554271_a_at Unknown Other 2.582 Chromosome 11 open reading C11ORF75219806_s_at Unknown Other 3.914 frame 75 Chromosome 3 open readingC3ORF70 242447_at Unknown Other 2.705 frame 70 Coiled-coil domainCCDC109B 218802_at Unknown Other 2.881 containing 109B Contactin 1 CNTN1211203_s_at Plasma Enzyme 3.684 Membrane Contactin 1 CNTN1 227202_atPlasma Enzyme 3.184 Membrane Contactin 1 CNTN1 227209_at Plasma Enzyme3.188 Membrane Copine VIII CPNE8 228365_at Unknown Other −3.355 CopineVIII CPNE8 241706_at Unknown Other −2.736 CUG triplet repeat, CUGBP2202157_s_at Nucleus Other 3.588 RNA binding protein 2 Cyclin-dependentkinase 2 CDK2 204252_at Nucleus Kinase 2.744 Cysteine and glycine-richCSRP2 207030_s_at Nucleus Other 2.487 protein 2 Cysteine andglycine-rich CSRP2 211126_s_at Nucleus Other 2.109 protein 2 DEP domaincontaining 1 DEPDC1 220295_x_at Unknown Other 2.795 Diaphanous homolog 3DIAPH3 232596_at Cytoplasm Enzyme 2.434 Dihydropyrimidine DPYD 204646_atCytoplasm Enzyme 4.248 dehydrogenase Docking protein 5 DOK5 214844_s_atPlasma Other 4.56 Membrane Dynein, light chain, DYNLT3 203303_atCytoplasm Other 3.449 Tctex-type 3 Elastin microfibril EMILIN2224374_s_at Extracellular Other 2.603 interfacer 2 Space Ets variant 1ETV1 206501_x_at Nucleus Transcription 2.578 regulator Ets variant 1ETV1 217053_x_at Nucleus Transcription 2.499 regulator Ets variant 1ETV1 217061_s_at Nucleus Transcription 2.557 regulator Family withsequence FAM101B 226876_at Unknown Other 2.399 similarity 101, member BFibroblast growth factor FGFR2 203638_s_at Plasma Kinase 2.306 receptor2 Membrane Fibroblast growth factor FGFR2 203639_s_at Plasma Kinase3.239 receptor 2 Membrane Fibroblast growth factor FGFR2 208228_s_atPlasma Kinase 2.151 receptor 2 Membrane Fibronectin type III domainFNDC5 226096_at Unknown Other 2.054 containing 5 Fibulin 1 FBLN1201787_at Extracellular Other 2.801 Space Fibulin 1 FBLN1 202994_s_atExtracellular Other 2.378 Space Fibulin 1 FBLN1 202995_s_atExtracellular Other 3.733 Space G protein-coupled receptor 177 GPR177221958_s_at Unknown Other 2.389 G protein-coupled receptor 177 GPR177228950_s_at Unknown Other 2.622 G-2 and S-phase expressed 1 GTSE1204317_at Cytoplasm Other 3.334 Gametocyte specific factor 1 GTSF1227711_at Unknown Other 6.407 Guanine nucleotide binding GNAI1 227692_atPlasma Enzyme 3.123 protein (G protein), alpha Membrane inhibitingactivity polypeptide 1 Guanine nucleotide binding GNB4 225710_at PlasmaEnzyme 6.066 protein (G protein), beta Membrane polypeptide 4 Guaninenucleotide binding GNG11 204115_at Plasma Enzyme 4.251 protein (Gprotein), gamma 11 Membrane Guanine nucleotide binding GNG12 212294_atPlasma Enzyme 2.315 protein (G protein), gamma 12 Membrane Guaninenucleotide binding GNG12 222834_s_at Plasma Enzyme 3.403 protein (Gprotein), gamma 12 Membrane Hepatocyte growth factor HGF 209960_atExtracellular Growth 2.939 (hepapoietin A) Space factor Hypotheticalprotein LOC100289109 216189_at Unknown Other −3.256 LOC100289109Hypothetical protein LOC144571 LOC144571 1564139_at Unknown Other −2.394Inositol 1,4,5-triphosphate ITPRIP 225582_at Unknown Other 3.522receptor interacting protein Interleukin 17B IL17B 220273_atExtracellular Cytokine −4.735 Space KDEL (Lys-Asp-Glu-Leu) KDELC2225128_at Unknown Other 3.003 containing 2 KIAA0125 KIAA0125 206478_atUnknown Other −8.436 KIT ligand KITLG 226534_at Extracellular Growth2.59 Space factor LY6/PLAUR domain LYPD6 227764_at Extracellular Other2.64 containing 6 Space Lysophosphatidic acid LPAR1 204036_at PlasmaReceptor 3.151 receptor 1 Membrane MAB-21-like 2 MAB21L2 210303_atUnknown Other 3.817 Minichromosome maintenance MCM10 223570_at NucleusOther 2.249 complex component 10 Moesin MSN 200600_at Plasma Other 5.842Membrane Monoamine oxidase A MAOA 204388_s_at Cytoplasm Enzyme 4.029Monoamine oxidase A MAOA 204389_at Cytoplasm Enzyme 3.355 Monoamineoxidase A MAOA 212741_at Cytoplasm Enzyme 4.157 MYC induced nuclearantigen MINA 213188_s_at Nucleus Other 4.019 MYC induced nuclear antigenMINA 213189_at Nucleus Other 4.636 Myosin VI MYO6 203216_s_at CytoplasmOther 2.406 Myosin VIIA and Rab MYRIP 214156_at Cytoplasm Other 2.805interacting protein Neuronal PAS domain protein 4 NPAS4 1554299_atNucleus Transcription −2.515 regulator Paternally expressed 3 PEG3209242_at Nucleus Kinase 5.896 Paternally expressed 3 PEG3 209243_s_atNucleus Kinase 4.556 Phosphoprotein associated with PAG1 225626_atPlasma Other 2.193 glycosphingolipid microdomains 1 Membrane Poliovirusreceptor-related 3 PVRL3 213325_at Plasma Other 2.325 Membrane Polo-likekinase 2 PLK2 201939_at Nucleus Kinase 4.93 Polymerase (DNA directed),POLA2 204441_s_at Nucleus Enzyme 2.198 alpha 2 (70 kD subunit) Prolactinreleasing hormone PRLHR 231805_at Plasma Receptor 3.19 receptor MembraneProstaglandin reductase 1 PTGR1 228824_s_at Cytoplasm Enzyme 2.398Prostaglandin reductase 1 PTGR1 231897_at Cytoplasm Enzyme 2.33Protease, serine, 12 PRSS12 205515_at Extracellular Peptidase 5.146(neurotrypsin, motopsin) Space Protein phosphatase 1, regulatory PPP1R3C204284_at Cytoplasm Phosphatase 2.827 (inhibitor) subunit 3C Proteintyrosine phosphatase, PTPRK 203038_at Plasma Phosphatase 3.451 receptortype, K Membrane RAB32, member RAS oncogene RAB32 204214_s_at CytoplasmOther 2.192 family RELT-like 1 RELL1 226430_at Unknown Other 2.88 RhoGTPase activating protein 24 ARHGAP24 223422_s_at Cytoplasm Other 3.043Rho guanine nucleotide ARHGEF3 218501_at Cytoplasm Other 2.473 exchangefactor (GEF) 3 Ring finger protein 182 RNF182 230720_at Unknown Other4.746 RNA binding protein with multiple RBPMS 209487_at Unknown Other2.329 splicing RNA binding protein with multiple RBPMS 209488_s_atUnknown Other 3.292 splicing Secreted protein, acidic, cysteine- SPARC200665_s_at Extracellular Other 4.919 rich (osteonectin) Space Secretedprotein, acidic, cysteine- SPARC 212667_at Extracellular Other 4.58 rich(osteonectin) Space Shugoshin-like 2 SGOL2 230165_at Nucleus Other 3.108Similar to hCG2031213 LOC728052 1558795_at Unknown Other 3.402 Solutecarrier family 1 (glial high SLC1A2 225491_at Plasma Transporter −4.354affinity glutamate transporter), Membrane member 2 Solute carrier family43, SLC43A3 210692_s_at Extracellular Other 3.126 member 3 Space Solutecarrier family 43, SLC43A3 213113_s_at Extracellular Other 2.804 member3 Space Solute carrier family 44, SLC44A5 1569112_at Unknown Other 3.923member 5 Solute carrier family 44, SLC44A5 235763_at Unknown Other 4.439member 5 Solute carrier family 7 (cationic SLC7A2 225516_at PlasmaTransporter 3.23 amino acid transporter, y+ Membrane system), member 2Sortilin-related VPS10 domain SORCS1 1556891_at Plasma Transporter−2.424 containing receptor 1 Membrane SPC25, NDC80 kinetochore SPC25209891_at Unknown Other 2.634 complex component, homolog ST8alpha-N-acetyl-neuraminide ST8SIA4 230836_at Cytoplasm Enzyme 2.705alpha-2,8-sialyltransferase 4 Structural maintenance of SMC2 204240_s_atNucleus Transporter 2.442 chromosomes 2 Structural maintenance of SMC6218781_at Nucleus Other 3.327 chromosomes 6 Structural maintenance ofSMC6 236535_at Nucleus Other 3.44 chromosomes 6 Syndecan 2 SDC2212154_at Plasma Other 2.367 Membrane Syndecan 2 SDC2 212158_at PlasmaOther 2.098 Membrane Thrombospondin 2 THBS2 203083_at ExtracellularOther −2.81 Space Thyroid hormone receptor TRIP10 202734_at CytoplasmOther 2.663 interactor 10 Tissue factor pathway inhibitor 2 TFPI2209278_s_at Extracellular Other 4.666 Space Transcription factor 7-like1 (T- TCF7L1 221016_s_at Nucleus Transcription 2.835 cell specific,HMG-box) regulator Transmembrane phosphatase TPTE 220205_at PlasmaPhosphatase 6.04 with tensin homology Membrane Transmembrane protein 178TMEM178 229302_at Unknown Other 3.194 Transmembrane protein 35 TMEM35219685_at Unknown Other 2.879 Tumor necrosis factor, alpha- TNFAIP8210260_s_at Cytoplasm Other 2.389 induced protein 8 Twist homolog 1TWIST1 213943_at Nucleus Transcription 2.573 regulator Zinc fingerprotein 521 ZNF521 226676_at Nucleus Other 2.382 Zinc finger protein 521ZNF521 226677_at Nucleus Other 2.943 Zinc finger protein 814 ZNF8141556204_a_at Unknown Other −2.505 The log ratio represent log₂ valueswhere 0.585 is log₂(1.5) which is a 1.5-fold difference, 1 is log₂(2)which is a 2-fold difference, 1.584 is log₂(3) which is a 3-folddifference, 2 is log₂(4) which is a 4-fold difference, 2.321 is log₂(5)which is a 5-fold difference, 2.584 is log₂(6) which is a 6-folddifference, 2.807 is log₂(7) which is a 7-fold difference, 3 is log₂(8)which is a 8-fold difference, 3.169 is log₂(9) which is a 9-folddifference, and 3.321 is log₂(10) which is a 10-fold difference.

Using IPA Core Analysis, 73 of the 79 genes were mapped to 5 differentprotein networks (Table 15). Based on overlapping gene mapping, 73 ofthe 79 genes that were up-regulated could be associated with two majorprotein network groups, named A and B (Table 15). Networks 1, 2 and 5belong to Group A, and Networks 3 and 4 belong to Group B. Similarly,all down-regulated genes were all associated with one network, named C(Table 15).

Group A genes include the FGFR2 receptor which has been shown tofunction in the BoNT/A intoxication process as a receptor for BoNT/A andare as follows: genes exhibiting a 1.5-fold or more increase inexpression levels in the H1 and BB10 cell lines as compared to theirexpression in cells from the 2D6 cell line, ADAMTS9, ARHGAP24, ARHGEF3,ASCL1, BARD1, CD9, CDK2, CSRP2, CTSL2, DIAPH3, DOK5, DYNLT3, EMILIN2,ETV1, FBLN1, FGFR2, GNAI1, GNB4, GNG11, GNG12, HGF, KITLG, LPAR1, MCM10,MSN, PAG1, PEG3, PLK2, POLA2, PPP1R3C, PTPRK, RAB32, SDC2, SLC43A3,SLC7A2, SMC6, SPARC, SPC25, ST8SIA4, TCF7L1, TFPI2, TMEM35, TNFAIP8,TPTE, TRIP10, and TWIST1.

Group B genes are as follows: genes exhibiting a 1.5-fold or moreincrease in expression levels in the H1 and BB10 cell lines as comparedto their expression in cells from the 2D6 cell line, ACOT9, BASP1,C11ORF75, CCDC109B, CDCA7L, CLSTN2, CNTN1, CUGBP2, DEPDC1, DPYD,FAM101B, FNDC5, GTSE1, MAOA, MINA, MYO6, MYRIP, PLK2, PRLHR, PVRL3,RBPMS, SGOL2, SMC2, TFP12, TMEM178, and ZNF521.

Group C genes are as follows: genes exhibiting a 1.5-fold or moredecrease in expression levels in the H1 and BB10 cell lines as comparedto their expression in cells from the 2D6 cell line, ANK2, CPNE8, CREB5,IL17B, KIAA0125, LOC100289109, LOC144571, NPAS4, SLC1A2, SORCS1, THBS2,and/or ZNF814.

The remaining networks identified did not share common genes between anyother network. Individually, however, genes exhibiting a 1.5-fold ormore increase in expression levels in the H1 and BB10 cell lines ascompared to their expression in cells from the 2D6 cell line were asfollows: C3ORF70 (Network 6), MAB21L2 (Network 7), PRSS12 (Network 8),CENPL (Network 9), GPR177 (Network 10), and PTGR1 (Network 11).

TABLE 15 IPA Core Analysis of Genes JMP Gene Expression Profile NetworkGroup Proteins in Network pvalue Number Increased Decreased 1 A ADAMTS9,AKT, ASCL1, CDK2, 48 22 ADAMTS9, — CSRP2, CTSL2, DOK5, E2F, ASCL1, CDK2,FBLN1, FSH, GNAI1, GNG11, CSRP2, CTSL2, hCG, HGF, KITLG, LPAR1, DOK5,FBLN1, MAPK, MCM10, MEK, MSN, GNAI1, GNG11, NFκB, P38, MAPK, PAG1,PDGFBB, HGF, KITLG, PEG3, PLK2, RAC, RAS, LPAR1, SDC2, SPARC, TFPI2,TGFβ, MCM10, MSN, TNFAIP8, TRIP10, VEGF PAG1, PEG3, PLK2, SDC2, SPARC,TFPI2, TNFAIP8, TRIP10 2 A CASP3, Caspase 3/7, CCL6, 35 17 DIAPH3, —DIAPH3, DYNLT3, EMILIN2, DYNLT3, EPM2A, ETV1, EYA2, FAM3B, EMILIN2,ETV1, FASTK, GNAI1, GNB4, GNG10, GNAI1, GNB4, GNG11, GNG12, GSTM2, HK1,GNG11, IGFBP3, IL13, PLCE1, POLA2, GNG12, PPP1R3C, PTPRK, PTPRZ1, POLA2,RAB32, SLC43A3, SLC7A2, PPP1R3C, SRC, ST8SIA4, TNF, TPTE, PTPRK, RAB32,TWIST1 SLC43A3, SLC7A2, ST8SIA4, TPTE, TWIST1 3 B ACOT9, AR, ATXN7,BASP1, 26 14 ACOT9, BASP1, — CCNE2, CCNK, CLSTN2, CLSTN2, CNTN1, CUGBP2,DGKA, CNTN1, FNDC5, GTSE1, IL4, MAOA, CUGBP2, MSH2, NOVA1, NF1, PICK1,FNDC5, PLK2, PNRC1, POM121, GTSE1, MAOA, PRLHR, PTP4A1, PTP4A2, PLK2,PRLHR, PVRL1, PVRL3, PVRL4, PVRL3, RBPMS, SERPINB5, SMARCA4, RBPMS,TFP12, SPN, TFP12, TP53, ZNF521 ZNF521 4 B ARHGAP17, AXL, C11ORF75, 2413 C11ORF75, — CAPN6, CCDC109B, CDCA7L, CCDC109B, DEPDC1, DPYD, EIF4E,CDCA7L, FAM101B, GRB2, HNRNPF, DEPDC1, HTATIP2, MAOA, MINA, DPYD,MIR124, MIRLET7A1, MYC, FAM101B, MYO6, MYRIP, NCAPD3, NF2, MAOA, MINA,PPP2R5A, PPP2R5D, MYO6, MYRIP, PPP2R5E, RPS16, RPS23, SGOL2, SMC2,SGOL2, SLC25A12, SMC2, TMEM178 SMC4, TMEM178, TUSC2, TXNIP, ZBTB16 5 AARHGAP24, ARHGEF, 17 10 ARHGAP24, — ARHGEF3, BARD1, CD9, CD53, ARHGEF3,CTNNB1, DUSP3, ERK, ERM, BARD1, CD9, FGF9, FGF18, FGF21, FGF23, FGFR2,LPAR1, FGFR2, IL1, IL6, IL17RD, SMC6, SPC25, Integrinα6β1, KLB, LEF/TCF,TCF7L1, LPAR1, MIRN297-2, PI3K, RAS TMEM35 homolog, RNAPOLII, SLC26A2,SMC6, SPC25, SREBF1, TACC1, TCF7L1, TMEM35, TPT1, WISP1 6 — C3ORF70,MIR31 2 1 C3ORF70 — 7 — MAB21L2, MIRN294 2 1 MAB21L2 — 8 — AGPN, PRSS122 1 PRSS12 — 9 — CENPL, MIRN340 2 1 CENPL — 10 — GPR177, MIRN324 2 1GPR177 — 11 — ADH, PTGR1 2 1 PTGR1 — 12 C ANK2, ATP2A2, BDNF, CEBPG, 228 — ANK2, CPNE8, CPNE8, CREB5, GRM3, GRM5, CREB5, IL17B, IGFBP2, IL17B,ITPR, ITPR1, KIAA0125, K+, L1CAM, MIRN330, LOC100289109, Neurotrophin,NFE2L1, NGFR, LOC144571, NME1, NPAS4, Pro-inflammatory NPAS4, Cytokine,PSEN1, SCN2A, SLC1A2, SCN3A, SCNN1B, SLC1A2, SORCS1, SLC8A1, SORCS1,SORT1, THBS2, ZNF814 TGFA, THBS2, TNF, TNFAIP6

Computer analysis was performed as in Example V, except that the list ofprobes reflects genes that were greater than 1.5-fold differentialexpressed in the H1 and the BB10 cell lines when compared to both the2D6 and SiMa parental cell lines. When the p-value cut off was set to0.05, this analysis identified 439 gene probes that could be classifiedto 369 genes (Table 16).

TABLE 16 Genes Identified using IPA Core Analysis Gene Log Gene NameSymbol Probe ID. No. Location Type Ratio 6-phosphofructo-2- PFKFB3202464_s_at Cytoplasm kinase 1.811 kinase/fructose-2,6- biphosphatase 3achaete-scute complex homolog 1 ASCL1 209985_s_at Nucleus transcription2.429 regulator achaete-scute complex homolog 1 ASCL1 209987_s_atNucleus transcription 2.097 regulator achaete-scute complex homolog 1ASCL1 209988_s_at Nucleus transcription 1.446 regulator achaete-scutecomplex homolog 1 ASCL1 213768_s_at Nucleus transcription 1.578regulator activated leukocyte cell adhesion ALCAM 1569362_at Plasmaother 2.612 molecule Membrane activated leukocyte cell adhesion ALCAM201951_at Plasma other 1.774 molecule Membrane activated leukocyte celladhesion ALCAM 201952_at Plasma other 1.521 molecule Membrane acyl-CoAthioesterase 1 ACOT1 202982_s_at Cytoplasm enzyme −2.875 acylphosphatase2, muscle type ACYP2 206833_s_at unknown enzyme −1.15 additional sexcombs like 3 ASXL3 233536_at unknown other 1.572 adenosine deaminase,RNA- ADARB1 203865_s_at Nucleus enzyme −2.474 specific, B1 adenylatecyclase 1 ADCY1 213245_at Plasma enzyme −1.935 Membraneadipocyte-specific adhesion ASAM 228082_at Plasma other −2.802 moleculeMembrane ADP-ribosylation factor 1 ARF1 1565651_at Cytoplasm enzyme1.063 ADP-ribosylation factor-like 17A ARL17A 243899_at unknown other0.741 anaphase promoting complex ANAPC7 225554_s_at unknown other 0.679subunit 7 ankyrin repeat and BTB (POZ) ABTB1 229164_s_at Cytoplasmtranslation −0.656 domain containing 1 regulator ankyrin repeat domain50 ANKRD50 225731_at unknown other −0.654 ankyrin repeat domain 50ANKRD50 225735_at unknown other −1.114 aspartate beta-hydroxylase ASPHD1214993_at unknown other −0.763 domain containing 1 ataxin 2-bindingprotein 1 A2BP1 1553422_s_at Cytoplasm other 1.453 ataxin 2-bindingprotein 1 A2BP1 221217_s_at Cytoplasm other 1.497 ATPase, Ca++transporting, ATP2B3 207026_s_at Plasma transporter −1.248 plasmamembrane 3 Membrane ATPase, Ca++ transporting, ATP2B3 242036_x_at Plasmatransporter −1.777 plasma membrane 3 Membrane basonuclin 2 BNC2220272_at Nucleus other 2.466 basonuclin 2 BNC2 230722_at Nucleus other1.641 βγcrystallin domain containing 3 CRYBG3 214030_at unknown other4.74 β-site APP-cleaving enzyme 1 BACE1 217904_s_at Cytoplasm peptidase−1.57 brain abundant, membrane BASP1 202391_at Plasma other 1.146attached signal protein 1 Membrane bromodomain and WD repeat BRWD1225446_at Nucleus transcription −1.018 domain containing 1 regulatorbruno-like 4, RNA binding BRUNOL4 238966_at Nucleus translation −0.81protein regulator butyrophilin, subfamily 3, BTN3A3 38241_at unknownother −1.108 member A3 C2 calcium-dependent domain C2CD2 212875_s_atunknown other −1.27 containing 2 C2 calcium-dependent domain C2CD4A241031_at unknown other −2.615 containing 4A cadherin 12, type 2(N-cadherin CDH12 207149_at Plasma other −1.408 2) Membranecalcium/calmodulin-dependent CAMK2N2 230706_s_at Nucleus other −0.692protein kinase II inhibitor 2 calmin (calponin-like, CLMN 213839_atCytoplasm other −1.911 transmembrane) calpain 2, (m/II) large subunitCAPN2 208683_at Cytoplasm peptidase −1.429 CAP, adenylate cyclase- CAP2212551_at Plasma other −1.974 associated protein, 2 Membrane CAP,adenylate cyclase- CAP2 212554_at Plasma other −3.68 associated protein,2 Membrane carboxypeptidase, vitellogenic- CPVL 208146_s_at unknownpeptidase 1.708 like CART prepropeptide CARTPT 206339_at Extracellularother −4.676 Space Cas-Br-M (murine) ecotropic CBLB 209682_at Nucleusother 1.032 retroviral transforming sequence b CCR4-NOT transcriptionCNOT6L 227119_at Cytoplasm other −0.785 complex, subunit 6-like CD302molecule CD302 203799_at Plasma receptor −1.518 Membrane CD9 moleculeCD9 201005_at Plasma other 1.415 Membrane CDP-diacylglycerol synthaseCDS1 226185_at Cytoplasm enzyme −1.74 (phosphatidatecytidylyltransferase) 1 cell division cycle 25 homolog B CDC25B201853_s_at Nucleus phosphatase 1.453 (S. pombe) chemokine (C—X—C motif)CXCR4 209201_x_at Plasma receptor 4.135 receptor 4 Membrane chemokine(C—X—C motif) CXCR4 211919_s_at Plasma receptor 3.957 receptor 4Membrane chemokine (C—X—C motif) CXCR4 217028_at Plasma receptor 3.176receptor 4 Membrane chloride channel 3 CLCN3 201733_at Plasma ionchannel −0.908 Membrane chloride channel 3 CLCN3 201734_at Plasma ionchannel −0.987 Membrane cholinergic receptor, nicotinic, CHRNA7210123_s_at Plasma receptor 3.098 alpha 7 Membrane chromosome 1 openreading C1ORF21 221272_s_at unknown other −0.74 frame 21 chromosome 1open reading C1ORF21 223127_s_at unknown other −0.748 frame 21chromosome 1 open reading C1ORF43 1555225_at unknown other 1.604 frame43 chromosome 10 open reading C10ORF58 224435_at Extracellular other0.918 frame 58 Space chromosome 12 open reading C12ORF49 218867_s_atunknown other 0.692 frame 49 chromosome 16 open reading C16ORF52230721_at unknown other −0.87 frame 52 chromosome 20 open readingC20ORF7 227160_s_at unknown other 0.624 frame 7 chromosome 21 openreading C21ORF57 227421_at unknown other −1.086 frame 57 chromosome 21open reading C21ORF57 239208_s_at unknown other −0.657 frame 57chromosome 3 open reading C3ORF23 1555905_a_at Cytoplasm other −2.797frame 23 chromosome 3 open reading C3ORF23 1555906_s_at Cytoplasm other−1.502 frame 23 chromosome 3 open reading C3ORF23 241666_at Cytoplasmother −1.017 frame 23 chromosome 9 open reading C9ORF150 227443_atunknown other −1.773 frame 150 claudin 12 CLDN12 223249_at Plasma other−0.766 Membrane COBW domain containing 1 CBWD1 226193_x_at unknown other0.849 coiled-coil domain containing 3 CCDC3 223316_at Cytoplasm other3.357 collagen, type VI, alpha 1 COL6A1 213428_s_at Extracellular other−1.638 Space cripto, FRL-1, cryptic family 1 CFC1 223753_s_atExtracellular other −2.834 Space cripto, FRL-1, cryptic family 1 CFC1236724_at Extracellular other −1.636 Space CSE1 chromosome segregationCSE1L 210765_at Nucleus transporter 1.581 1-like CUG triplet repeat, RNAbinding CUGBP2 202156_s_at Nucleus other 1.318 protein 2 CUG tripletrepeat, RNA binding CUGBP2 202157_s_at Nucleus other 2.156 protein 2 CUGtriplet repeat, RNA binding CUGBP2 202158_s_at Nucleus other 2.539protein 2 cyclic nucleotide gated channel CNGA3 207261_at Plasma ionchannel −3.182 alpha 3 Membrane cyclin M1 CNNM1 220166_at Plasma other−1.214 Membrane cyclin-dependent kinase inhibitor CDKN2A 209644_x_atNucleus transcription −1.277 2A (melanoma, p16, inhibits regulator CDK4)cylindromatosis (turban tumor CYLD 213295_at Nucleus transcription−0.815 syndrome) regulator cytochrome P450, family 2, CYP2E1 1431_atCytoplasm enzyme −4.466 subfamily E, polypeptide 1 cytochrome P450,family 2, CYP2E1 209975_at Cytoplasm enzyme −3.946 subfamily E,polypeptide 1 cytochrome P450, family 2, CYP2E1 209976_s_at Cytoplasmenzyme −3.429 subfamily E, polypeptide 1 cytoglobin CYGB 1553572_a_atCytoplasm transporter −4.921 cytokine-like 1 CYTL1 219837_s_atExtracellular cytokine −6.33 Space dapper, antagonist of beta- DACT1219179_at Cytoplasm other 1.682 catenin, homolog 1 dCMP deaminase DCTD201572_x_at unknown enzyme −0.649 DEAH (Asp-Glu-Ala-His) box DHX35218579_s_at unknown enzyme 0.735 polypeptide 35 deleted in azoospermia 1DAZ1 207909_x_at Nucleus translation 6.92 regulator deleted inazoospermia 1 DAZ1 207912_s_at Nucleus translation 5.558 regulatordeleted in azoospermia 1 DAZ1 208281_x_at Nucleus translation 5.258regulator deleted in azoospermia 1 DAZ1 208282_x_at Nucleus translation7.008 regulator deleted in azoospermia 1 DAZ1 216351_x_at Nucleustranslation 3.596 regulator deleted in azoospermia 1 DAZ1 216922_x_atNucleus translation 5.185 regulator deleted in lymphocytic leukemiaDLEU2 216870_x_at unknown other 1.233 2 (non-protein coding) delta-like3 (Drosophila) DLL3 219537_x_at Extracellular other 0.779 Space DNAsegment on chromosome 4 D4S234E 209569_x_at Cytoplasm other −1.247(unique) 234 expressed sequence DNA segment on chromosome 4 D4S234E209570_s_at Cytoplasm other −1.488 (unique) 234 expressed sequence DNAsegment on chromosome 4 D4S234E 213533_at Cytoplasm other −1.507(unique) 234 expressed sequence dopamine beta-hydroxylase DBH 206450_atCytoplasm enzyme 1.311 (dopamine beta- monooxygenase) doublecortin-likekinase 1 DCLK1 215303_at Cytoplasm kinase −1.62 dual specificityphosphatase 16 DUSP16 224832_at Nucleus phosphatase −0.918 echinodermmicrotubule EML1 204797_s_at Cytoplasm other 2.668 associated proteinlike 1 ectodermal-neural cortex (with ENC1 201340_s_at Nucleus peptidase2.445 BTB-like domain) ectodermal-neural cortex (with ENC1 201341_atNucleus peptidase 1.91 BTB-like domain)electron-transferring-flavoprotein ETFDH 33494_at Cytoplasm enzyme−0.807 dehydrogenase ELL associated factor 2 EAF2 219551_at Nucleustranscription 0.693 regulator ELOVL family member 7, ELOVL7 227180_atunknown other 2.644 elongation of long chain fatty acids endoplasmicreticulum ERAP1 214012_at Extracellular peptidase −1.885 aminopeptidase1 Space enhancer of zeste homolog 2 EZH2 203358_s_at Nucleustranscription 0.94 regulator ephrin-A5 EFNA5 214036_at Plasma kinase2.041 Membrane ephrin-A5 EFNA5 227955_s_at Plasma kinase 1.84 Membraneephrin-A5 EFNA5 233814_at Plasma kinase 1.805 Membrane ephrin-B3 EFNB3205031_at Plasma kinase −0.816 Membrane eukaryotic translationinitiation EIF3C 236700_at Cytoplasm translation 1.378 factor 3, subunitC regulator exophilin 5 EXPH5 214734_at unknown other 3.973 exosomecomponent 6 EXOSC6 231916_at Nucleus other 1.057 family with sequencesimilarity FAM162B 228875_at unknown other −0.743 162, member B familywith sequence similarity FAM165B 228239_at Plasma other −0.822 165,member B Membrane family with sequence similarity FAM181B 231430_atunknown other 0.794 181, member B family with sequence similarityFAM19A4 242348_at Extracellular other 1.282 19 (chemokine (C-Cmotif)-like), Space member A4 family with sequence similarity FAM46A224973_at unknown other −1.509 46, member A family with sequencesimilarity 7, FAM7A3 243356_at unknown other 3.495 member A3 farupstream element (FUSE) FUBP3 239193_at Nucleus transcription 1.192binding protein 3 regulator F-box and WD repeat domain FBXW7 229419_atNucleus transcription −0.921 containing 7 regulator fibroblast growthfactor 13 FGF13 205110_s_at Extracellular growth factor −3.522 Spacefollistatin-like 1 FSTL1 208782_at Extracellular other −1.275 Spaceforkhead box O6 FOXO6 239657_x_at Nucleus other −1.512 frizzled homolog5 FZD5 221245_s_at Plasma receptor 2.347 Membrane FYVE, RhoGEF and PHdomain FGD5 226985_at Cytoplasm other −1.952 containing 5 Gprotein-coupled receptor 123 GPR123 239221_at Plasma receptor −1.302Membrane G protein-coupled receptor 125 GPR125 210473_s_at Plasmareceptor 0.714 Membrane galanin prepropeptide GAL 207466_atExtracellular other −1.434 Space galanin prepropeptide GAL 214240_atExtracellular other −3.128 Space gastrin-releasing peptide GRP 206326_atExtracellular growth factor 4.698 Space GDNF family receptor alpha 2GFRA2 205721_at Plasma receptor 1.847 Membrane GDNF family receptoralpha 2 GFRA2 205722_s_at Plasma receptor 2.265 Membrane glutamatereceptor, metabotropic 5 GRM5 214217_at Plasma receptor −2.231 Membraneglutathione peroxidase 7 GPX7 213170_at Cytoplasm enzyme −2.692glutathione synthetase GSS 211630_s_at Cytoplasm enzyme 0.599glycosyltransferase 25 domain GLT25D2 209883_at unknown other −1.223containing 2 GNAS complex locus GNAS 214157_at Plasma enzyme −1.794Membrane growth associated protein 43 GAP43 204471_at Plasma other 0.869Membrane growth associated protein 43 GAP43 216963_s_at Plasma other1.151 Membrane guanine nucleotide binding GNAI1 209576_at Plasma enzyme2.225 protein (G protein), alpha Membrane inhibiting activitypolypeptide 1 guanine nucleotide binding GNAI1 227692_at Plasma enzyme2.111 protein (G protein), alpha Membrane inhibiting activitypolypeptide 1 guanine nucleotide binding GNB4 225710_at Plasma enzyme4.695 protein (G protein), beta Membrane polypeptide 4 guaninenucleotide binding GNG11 204115_at Plasma enzyme 2.587 protein (Gprotein), gamma 11 Membrane guanylate cyclase 1, soluble, GUCY1A3221942_s_at Cytoplasm enzyme −1.522 alpha 3 guanylate cyclase 1,soluble, GUCY1A3 229530_at Cytoplasm enzyme −1.696 alpha 3 helicase,POLQ-like HELQ 228736_at Nucleus enzyme −1.28 HERPUD family member 2HERPUD2 236170_x_at unknown other −0.897 heterogeneous nuclear HNRNPR208765_s_at Nucleus other −0.64 ribonucleoprotein R hippocalcin-like 1HPCAL1 205462_s_at Cytoplasm other −1.551 hippocalcin-like 1 HPCAL1212552_at Cytoplasm other −1.387 histone cluster 1, H2ac HIST1H2AC215071_s_at Nucleus other −2.462 histone cluster 1, H2bd HIST1H2BD209911_x_at Nucleus other −2.036 histone cluster 1, H2bk HIST1H2BK209806_at Nucleus other −1.575 histone cluster 2, H2be HIST2H2BE202708_s_at Nucleus other −1.735 hypothetical LOC100130522 LOC100130522230477_at unknown other −0.928 hypothetical LOC26082 DKFZP434L187230861_at unknown other 0.968 hypothetical protein LOC100128844229110_at unknown other 2.106 LOC100128844 hypothetical proteinLOC643401 LOC340109 1557765_at unknown other 2.848 integrin, alpha 6ITGA6 201656_at Plasma other 2.822 Membrane integrin, alpha 6 ITGA6215177_s_at Plasma other 2.024 Membrane integrin, beta 5 ITGB5201125_s_at Plasma other 0.913 Membrane interleukin 10 receptor, betaIL10RB 209575_at Plasma transmembrane −1.444 Membrane receptor junctionmediating and JMY 226352_at Nucleus transcription −0.744 regulatoryprotein, p53 cofactor regulator K(lysine) acetyltransferase 2B KAT2B203845_at Nucleus transcription −0.731 regulator KDEL (Lys-Asp-Glu-Leu)KDELC2 225128_at unknown other 1.085 containing 2 kelch domaincontaining 1 KLHDC1 1552733_at unknown other −0.952 kelch repeat and BTB(POZ) KBTBD11 204301_at unknown other −0.733 domain containing 11kelch-like 13 (Drosophila) KLHL13 227875_at unknown other 0.902 KHdomain containing, RNA KHDRBS3 209781_s_at Nucleus other 1.082 binding,signal transduction associated 3 KH homology domain containing 1 KHDC1230055_at unknown other −1.656 KIAA1598 KIAA1598 221802_s_at unknownother −2.811 kinesin family member 16B KIF16B 232083_at Cytoplasm other1.688 lectin, galactoside-binding, LGALS3BP 200923_at Plasmatransmembrane −1.417 soluble, 3 binding protein Membrane receptorleukemia inhibitory factor LIFR 225575_at Plasma transmembrane −0.941receptor alpha Membrane receptor limb bud and heart development LBH221011_s_at Nucleus transcription 0.788 homolog (mouse) regulator LRP2binding protein LRP2BP 207797_s_at unknown other −1.365 lumican LUM201744_s_at Extracellular other 5.638 Space LYR motif containing 5 LYRM5225469_at unknown other −0.93 mab-21-like 1 MAB21L1 206163_at unknownother 2.722 mab-21-like 2 MAB21L2 210302_s_at unknown other 2.871mab-21-like 2 MAB21L2 210303_at unknown other 2.204 macrophagestimulating 1 MST1 216320_x_at Extracellular growth factor −1.012(hepatocyte growth factor-like) Space major facilitator superfamilyMFSD4 229254_at unknown other −1.752 domain containing 4 mannosidase,alpha, class 2A, MAN2A1 226538_at Cytoplasm enzyme 0.864 member 1mannosyl (alpha-1,3-)- MGAT4A 226039_at unknown enzyme −0.904glycoprotein beta-1,4-N- acetylglucosaminyltransferase, isozyme Amesenchyme homeobox 2 MEOX2 206201_s_at Nucleus transcription −4.213regulator microtubule-associated protein 9 MAP9 220145_at unknown other−0.7 microtubule-associated protein 9 MAP9 228423_at unknown other−1.108 microtubule-associated protein 9 MAP9 235550_at unknown other−0.863 mitochondrial ribosomal protein MRPS33 218654_s_at Cytoplasmother −0.609 S33 mitogen-activated protein kinase MAP3K5 203836_s_atCytoplasm kinase −1.159 kinase kinase 5 moesin MSN 200600_at Plasmaother 2.082 Membrane monoamine oxidase A MAOA 204388_s_at Cytoplasmenzyme 2.15 monoamine oxidase A MAOA 204389_at Cytoplasm enzyme 1.88monoamine oxidase A MAOA 212741_at Cytoplasm enzyme 2.048 monocyte tomacrophage MMD 244523_at Plasma other 1.045 differentiation-associatedMembrane multiple C2 domains, MCTP1 220122_at unknown other −1.597transmembrane 1 MYC induced nuclear antigen MINA 213188_s_at Nucleusother 1.391 MYC induced nuclear antigen MINA 213189_at Nucleus other1.641 myosin IB MYO1B 212365_at Cytoplasm other 0.71 myosin VI MYO6203216_s_at Cytoplasm other 1.104 myosin VI MYO6 210480_s_at Cytoplasmother 1.41 Na+/H+ exchanger domain NHEDC2 1564746_at Plasma other −1.462containing 2 Membrane N-acylethanolamine acid NAAA 214765_s_at Cytoplasmenzyme 0.911 amidase NADH dehydrogenase NDUFA4L2 218484_at unknownenzyme −3.507 (ubiquinone) 1 alpha subcomplex, 4-like 2 NADHdehydrogenase NDUFV3 226616_s_at Cytoplasm enzyme −0.875 (ubiquinone)flavoprotein 3, 10 kDa nebulette NEBL 203961_at Cytoplasm other −1.359neural precursor cell expressed, NEDD9 202150_s_at Nucleus other 2.41developmentally down-regulated 9 NIPA-like domain containing 3 NIPAL3225876_at unknown other −1.203 nitric oxide synthase 1 NOS1 239132_atCytoplasm enzyme 1.138 (neuronal) nitric oxide synthase 1 NOS1 240911_atCytoplasm enzyme 1.187 (neuronal) NLR family member X1 NLRX1 219680_atunknown other −1.541 non-protein coding RNA 171 NCRNA00171 215985_atunknown other −0.692 nuclear receptor coactivator 7 NCOA7 225344_atNucleus other −0.842 nucleosome assembly protein 1- NAP1L3 204749_atNucleus other −1.53 like 3 nudix (nucleoside diphosphate NUDT19235384_at Cytoplasm other −1.685 linked moiety X)-type motif 19olfactomedin 3 OLFM3 1554524_a_at Cytoplasm other −0.927 oxysterolbinding protein-like 3 OSBPL3 209626_s_at Cytoplasm other 2.083pecanex-like 2 PCNXL2 1554256_a_at unknown other −0.728 pellino homolog2 PELI2 219132_at Cytoplasm other 0.732 peptidase M20 domain PM20D2225421_at unknown other 1.6 containing 2 peptidylglycine alpha-amidatingPAM 202336_s_at Plasma enzyme −0.861 monooxygenase Membrane peripherinPRPH 213847_at Plasma other −1.417 Membrane PHD finger protein 20 PHF20235389_at Nucleus other 0.596 phosphatidic acid phosphatase PPAPDC1A236044_at unknown phosphatase −2.621 type 2 domain containing 1Aphosphatidylinositol glycan PIGH 209625_at Cytoplasm enzyme −0.597anchor biosynthesis, class H phosphoinositide-interacting HCG1776018232887_at unknown other −2.019 regulator of transient receptor potentialchannels phosphorylase kinase, alpha 1 PHKA1 229876_at Cytoplasm kinase−1.2 (muscle) plastin 3 (T isoform) PLS3 201215_at Cytoplasm other 1.068platelet-derived growth factor PDGFRB 202273_at Plasma kinase −1.373receptor, beta polypeptide Membrane pleiotrophin PTN 209466_x_atExtracellular growth factor −1.426 Space pleiotrophin PTN 211737_x_atExtracellular growth factor −2.929 Space pleiotropic regulator 1 PLRG1225194_at Nucleus transcription −0.701 regulator pleiotropic regulator 1PLRG1 227246_at Nucleus transcription −1.228 regulator plexin A2 PLXNA2213030_s_at Plasma other 0.946 Membrane plexin A4 PLXNA4 232317_atPlasma receptor 1.368 Membrane polo-like kinase 2 PLK2 201939_at Nucleuskinase 3.606 polycomb group ring finger 5 PCGF5 227935_s_at unknownother 0.791 polymerase (DNA directed), POLE 216026_s_at Nucleus enzyme1.053 epsilon potassium channel KCTD12 212188_at unknown ion channel3.396 tetramerisation domain containing 12 potassium channel KCTD12212192_at unknown ion channel 4.806 tetramerisation domain containing 12potassium large conductance KCNMA1 221584_s_at Plasma ion channel −2.389calcium-activated channel, Membrane subfamily M, alpha member 1potassium voltage-gated KCNQ2 205737_at Plasma ion channel −1.246channel, KQT-like subfamily, Membrane member 2 pregnancy-associatedplasma PAPPA 201982_s_at Extracellular peptidase −1.672 protein A,pappalysin 1 Space prostaglandin E receptor 2 PTGER2 206631_at Plasmareceptor −2.328 (subtype EP2), 53 kDa Membrane prostaglandin F2 receptorPTGFRN 224937_at Plasma other 2.301 negative regulator Membraneproteasome (prosome, PSMA7 216088_s_at Cytoplasm peptidase 0.658macropain) subunit, alpha type, 7 protein kinase, cAMP- PRKACB 235780_atCytoplasm kinase −2.17 dependent, catalytic, beta protein tyrosinephosphatase, PTPRD 214043_at Plasma phosphatase −1.16 receptor type, DMembrane protein tyrosine phosphatase, PTPRE 221840_at Plasmaphosphatase 3.044 receptor type, E Membrane protein tyrosinephosphatase, PTPRG 204944_at Plasma phosphatase 1.237 receptor type, GMembrane RAB35, member RAS oncogene RAB35 205461_at Cytoplasm enzyme0.737 family RAB6B, member RAS oncogene RAB6B 225259_at Cytoplasm enzyme−1.229 family Rac/Cdc42 guanine nucleotide ARHGEF6 209539_at Cytoplasmother 2.719 exchange factor (GEF) 6 RAN, member RAS oncogene RAN200750_s_at Nucleus enzyme 0.59 family RAS and EF-hand domain RASEF1553986_at unknown other 1.393 containing ras homolog gene family, RHOU223168_at Cytoplasm enzyme −1.154 member U regulator of G-proteinsignaling 5 RGS5 1555725_a_at Plasma other 4.038 Membrane regulator ofG-protein signaling 5 RGS5 209070_s_at Plasma other 3.729 Membraneregulator of G-protein signaling 5 RGS5 209071_s_at Plasma other 3.468Membrane regulator of G-protein signaling 5 RGS5 218353_at Plasma other3.65 Membrane RELT-like 1 RELL1 226430_at unknown other 1.214retinoblastoma-like 1 (p107) RBL1 1559307_s_at Nucleus other 0.959retrotransposon gag domain RGAG4 227823_at unknown other −1.775containing 4 Rho GTPase activating protein 6 ARHGAP6 206167_s_atCytoplasm other 2.809 ribosomal L1 domain containing 1 RSL1D1 213750_atCytoplasm other −0.816 RIMS binding protein 2 RIMBP2 238817_at Plasmaother 1.679 Membrane ring finger protein 13 RNF13 201780_s_at Nucleusother −0.707 ring finger protein 182 RNF182 230720_at unknown other2.696 ring finger protein 34 RNF34 219035_s_at Cytoplasm enzyme 0.85ring finger protein 41 RNF41 201962_s_at Cytoplasm other −0.591 RNApolymerase I transcription LOC94431 216908_x_at unknown other −0.674factor homolog pseudogene 1 sal-like 4 SALL4 229661_at Nucleus other1.586 salt-inducible kinase 1 SIK1 208078_s_at Cytoplasm kinase −1.403seizure related 6 homolog SEZ6L 207873_x_at Plasma other 0.866(mouse)-like Membrane seizure related 6 homolog SEZ6L 211894_x_at Plasmaother 0.797 (mouse)-like Membrane sema domain, transmembrane SEMA6A220454_s_at Plasma other 1.356 domain (TM), and cytoplasmic Membranedomain, (semaphorin) 6A serine peptidase inhibitor, Kunitz SPINT2210715_s_at Extracellular other −1.889 type, 2 Space SH3 domaincontaining, Ysc84- SH3YL1 204019_s_at unknown other −0.859 like 1SH3-domain GRB2-like 2 SH3GL2 205751_at Plasma enzyme −1.605 Membranesimilar to hCG2031213 LOC728052 1558795_at unknown other 2.239 similarto hCG2031213 LOC728052 1558796_a_at unknown other 1.887 singleimmunoglobulin and toll- SIGIRR 52940_at Plasma receptor −1.42interleukin 1 receptor (TIR) Membrane domain single-minded homolog 1SIM1 1556300_s_at Nucleus transcription 6.36 regulator single-mindedhomolog 1 SIM1 206876_at Nucleus transcription 5.737 regulator SLIT andNTRK-like family, SLITRK5 214930_at unknown other 4.094 member 5 SMG1homolog, LOC641298 244766_at unknown other 1.065 phosphatidylinositol3-kinase- related kinase pseudogene solute carrier family 12 SLC12A7218066_at Plasma transporter −1.258 (potassium/chloride Membranetransporters), member 7 solute carrier family 22, member SLC22A17218675_at Plasma transporter −0.724 17 Membrane solute carrier family35, member SLC35F3 229065_at unknown other −3.048 F3 solute carrierfamily 44, member 5 SLC44A5 1569112_at unknown other 2.738 solutecarrier family 44, member 5 SLC44A5 235763_at unknown other 1.845 solutecarrier family 7 (cationic SLC7A2 225516_at Plasma transporter 1.892amino acid transporter, y+ Membrane system), member 2 solute carrierorganic anion SLCO3A1 219229_at Plasma transporter 1.622 transporterfamily, member 3A1 Membrane solute carrier organic anion SLCO3A1227367_at Plasma transporter 1.045 transporter family, member 3A1Membrane sparc/osteonectin, cwcv and SPOCK2 202524_s_at Extracellularother −0.83 kazal-like domains proteoglycan Space (testican) 2 spermassociated antigen 6 SPAG6 210032_s_at Cytoplasm other 1.687 spermassociated antigen 6 SPAG6 210033_s_at Cytoplasm other 2.774spermatogenesis associated, SPATS2L 222154_s_at unknown other 0.907serine-rich 2-like sphingosine-1-phosphate S1PR3 228176_at Plasmareceptor 1.9 receptor 3 Membrane ST8 alpha-N-acetyl-neuraminide ST8SIA1210073_at Cytoplasm enzyme 1.114 alpha-2,8-sialyltransferase 1 ST8alpha-N-acetyl-neuraminide ST8SIA3 230262_at Cytoplasm enzyme −1.039alpha-2,8-sialyltransferase 3 stathmin-like 3 STMN3 222557_at Nucleusother 0.788 STEAP family member 3 STEAP3 218424_s_at Cytoplasmtransporter −1.827 steroidogenic acute regulatory STAR 204548_atCytoplasm transporter −1.093 protein stimulated by retinoic acid geneSTRA6 1569334_at Plasma other 1.359 6 homolog Membrane stimulated byretinoic acid gene STRA6 1569335_a_at Plasma other 1.167 6 homologMembrane storkhead box 2 STOX2 226822_at unknown other −0.854synaptojanin 1 SYNJ1 212990_at Cytoplasm phosphatase −0.624synaptotagmin XIII SYT13 226086_at unknown transporter −4.754 synuclein,alpha (non A4 SNCA 236081_at Cytoplasm other −0.935 component of amyloidprecursor) taxilin beta TXLNB 227834_at Cytoplasm other −2.226 teashirtzinc finger homeobox 3 TSHZ3 223392_s_at Nucleus transcription 1.472regulator teashirt zinc finger homeobox 3 TSHZ3 223393_s_at Nucleustranscription 0.962 regulator tetratricopeptide repeat domain TTC39C238480_at unknown other −0.824 39C TH1-like TH1L 220607_x_at Nucleusother 1.027 TH1-like TH1L 225006_x_at Nucleus other 0.884 TH1-like TH1L225261_x_at Nucleus other 0.883 TH1-like TH1L 225865_x_at Nucleus other0.904 thioredoxin reductase 1 TXNRD1 201266_at Cytoplasm enzyme 1.032THO complex 4 THOC4 226319_s_at Nucleus transcription 0.996 regulatorTIMP metallopeptidase inhibitor 3 TIMP3 201147_s_at Extracellular other5.151 Space TIMP metallopeptidase inhibitor 3 TIMP3 201148_s_atExtracellular other 3.637 Space TIMP metallopeptidase inhibitor 3 TIMP3201149_s_at Extracellular other 5.037 Space TIMP metallopeptidaseinhibitor 3 TIMP3 201150_s_at Extracellular other 4.312 Space tissuefactor pathway inhibitor 2 TFPI2 209277_at Extracellular other 1.592Space tissue factor pathway inhibitor 2 TFPI2 209278_s_at Extracellularother 2.326 Space TM2 domain containing 1 TM2D1 213882_at Plasmareceptor −0.769 Membrane transcription factor 7-like 2 (T- TCF7L2212761_at Nucleus transcription 1.005 cell specific, HMG-box) regulatortranscription factor 7-like 2 (T- TCF7L2 212762_s_at Nucleustranscription 1.224 cell specific, HMG-box) regulator transcriptionfactor 7-like 2 (T- TCF7L2 216035_x_at Nucleus transcription 1.072 cellspecific, HMG-box) regulator transcription factor 7-like 2 (T- TCF7L2216037_x_at Nucleus transcription 0.81 cell specific, HMG-box) regulatortranscription factor 7-like 2 (T- TCF7L2 216511_s_at Nucleustranscription 1.003 cell specific, HMG-box) regulator transducin-likeenhancer of split 3 TLE3 212770_at Nucleus other 0.976 transformer 2alpha homolog TRA2A 213593_s_at Nucleus other 1.12 transmembrane innerear TMIE 1553601_a_at unknown other −3.334 transmembrane protein 132CTMEM132C 232313_at unknown other 4.02 transmembrane protein 178 TMEM178229302_at unknown other 1.697 transmembrane protein 184C TMEM184C219074_at unknown other −0.779 transmembrane protein 5 TMEM5 204808_s_atPlasma other −1.074 Membrane transmembrane protein 59-like TMEM59L219005_at Cytoplasm other −0.845 tripartite motif-containing 29 TRIM29211002_s_at Cytoplasm transcription 2.005 regulator tripartitemotif-containing 36 TRIM36 219736_at Cytoplasm other 2.221 trophoblastglycoprotein TPBG 203476_at Plasma other 2.158 Membrane tumor suppressorcandidate 3 TUSC3 232770_at Extracellular enzyme −1.407 Space U2 smallnuclear RNA auxiliary U2AF1 232141_at Nucleus other 0.776 factor 1ubiquitin-conjugating enzyme E2 UBE2V1 201003_x_at Nucleus transcription0.663 variant 1 regulator urocortin UCN 206072_at Extracellular other−0.961 Space vacuolar protein sorting 29 VPS29 223026_s_at Cytoplasmtransporter 0.805 homolog v-erb-a erythroblastic leukemia ERBB4206794_at Plasma kinase −1.537 viral oncogene homolog 4 Membrane v-erb-aerythroblastic leukemia ERBB4 214053_at Plasma kinase −1.708 viraloncogene homolog 4 Membrane v-erb-b2 erythroblastic leukemia ERBB2216836_s_at Plasma kinase −0.698 viral oncogene homolog 2, Membraneneuro/glioblastoma derived oncogene homolog v-raf-1 murine leukemiaviral RAF1 1557675_at Cytoplasm kinase 0.696 oncogene homolog 1 WDrepeat and FYVE domain WDFY3 212602_at Cytoplasm enzyme −0.626containing 3 WD repeat and FYVE domain WDFY3 212606_at Cytoplasm enzyme−0.785 containing 3 zinc finger homeobox 4 ZFHX4 219779_at unknown other1.36 zinc finger homeobox 4 ZFHX4 241700_at unknown other 1.234 zincfinger protein 217 ZNF217 203739_at Nucleus transcription 1.096regulator zinc finger protein 503 ZNF503 227195_at Nucleus other 1.373zinc finger protein 641 ZNF641 226509_at unknown other −1.427 zincfinger protein 662 ZNF662 228538_at unknown other −3.352 zinc finger,CCCH-type with G ZGPAT 221848_at unknown other 0.991 patch domain

Computer analysis was performed as in Example V, except that the list ofprobes reflects genes that were greater than 1.5-fold differentialexpressed in the H1 cell line and the BB10 cell line when compared toboth the 2D6 and SiMa parental cell lines, and also a greater than1.5-fold differential expressed in the SiMa parental cell line whencompared to the 2D6 cell line. When the p-value cut off was set to 0.05,this analysis identified 94 gene probes that could be classified to 70genes (Table 17).

TABLE 17 Genes Identified using IPA Core Analysis Gene Log Gene NameSymbol Probe ID. No. Location Type Ratio achaete-scute complex ASCL1209985_s_at Nucleus transcription 2.429 homolog 1 regulatorachaete-scute complex ASCL1 209988_s_at Nucleus transcription 1.446homolog 1 regulator achaete-scute complex ASCL1 213768_s_at Nucleustranscription 1.578 homolog 1 regulator activated leukocyte cell ALCAM201952_at Plasma other 1.521 adhesion molecule Membrane ADP-ribosylationfactor 1 ARF1 1565651_at Cytoplasm enzyme 1.063 ADP-ribosylationfactor-like ARL17A 243899_at unknown other 0.741 17A ataxin 2-bindingprotein 1 A2BP1 1553422_s_at Cytoplasm other 1.453 ataxin 2-bindingprotein 1 A2BP1 221217_s_at Cytoplasm other 1.497 brain abundant,membrane BASP1 202391_at Plasma other 1.146 attached signal protein 1Membrane CAP, adenylate cyclase- CAP2 212551_at Plasma other −1.974associated protein, 2 Membrane CD9 molecule CD9 201005_at Plasma other1.415 Membrane CUG triplet repeat, RNA CUGBP2 202157_s_at Nucleus other2.156 binding protein 2 cyclic nucleotide gated CNGA3 207261_at Plasmaion channel −3.182 channel alpha 3 Membrane cyclin-dependent kinaseCDKN2A 209644_x_at Nucleus transcription −1.277 inhibitor 2A (melanoma,p16, regulator inhibits CDK4) ELL associated factor 2 EAF2 219551_atNucleus transcription 0.693 regulator eukaryotic translation initiationEIF3C 236700_at Cytoplasm translation 1.378 factor 3, subunit Cregulator family with sequence FAM162B 228875_at unknown other −0.743similarity 162, member B family with sequence FAM181B 231430_at unknownother 0.794 similarity 181, member B far upstream element (FUSE) FUBP3239193_at Nucleus transcription 1.192 binding protein 3 regulatorfibroblast growth factor 13 FGF13 205110_s_at Extracellular growthfactor −3.522 Space follistatin-like 1 FSTL1 208782_at Extracellularother −1.275 Space forkhead box O6 FOXO6 239657_x_at Nucleus other−1.512 guanine nucleotide binding GNAI1 227692_at Plasma enzyme 2.111protein (G protein), alpha Membrane inhibiting activity polypeptide 1guanine nucleotide binding GNB4 225710_at Plasma enzyme 4.695 protein (Gprotein), beta Membrane polypeptide 4 guanine nucleotide binding GNG11204115_at Plasma enzyme 2.587 protein (G protein), gamma 11 MembraneKDEL (Lys-Asp-Glu-Leu) KDELC2 225128_at unknown other 1.085 containing 2kelch-like 13 KLHL13 227875_at unknown other 0.902 KH domain containing,RNA KHDRBS3 209781_s_at Nucleus other 1.082 binding, signal transductionassociated 3 KH homology domain KHDC1 230055_at unknown other −1.656containing 1 leukemia inhibitory factor LIFR 225575_at Plasmatransmembrane −0.941 receptor alpha Membrane receptor mab-21-like 2MAB21L2 210303_at unknown other 2.204 mesenchyme homeobox 2 MEOX2206201_s_at Nucleus transcription −4.213 regulator moesin MSN 200600_atPlasma other 2.082 Membrane monoamine oxidase A MAOA 204388_s_atCytoplasm enzyme 2.15 monoamine oxidase A MAOA 204389_at Cytoplasmenzyme 1.88 monoamine oxidase A MAOA 212741_at Cytoplasm enzyme 2.048multiple C2 domains, MCTP1 220122_at unknown other −1.597 transmembrane1 MYC induced nuclear antigen MINA 213188_s_at Nucleus other 1.391 MYCinduced nuclear antigen MINA 213189_at Nucleus other 1.641 myosin VIMYO6 203216_s_at Cytoplasm other 1.104 phosphatidic acid PPAPDC1A236044_at unknown phosphatase −2.621 phosphatase type 2 domaincontaining 1A plastin 3 (T isoform) PLS3 201215_at Cytoplasm other 1.068pleiotrophin PTN 209466_x_at Extracellular growth factor −1.426 Spacepleiotrophin PTN 211737_x_at Extracellular growth factor −2.929 Spacepolo-like kinase 2 PLK2 201939_at Nucleus kinase 3.606 (Drosophila)potassium large conductance KCNMA1 221584_s_at Plasma ion channel −2.389calcium-activated channel, Membrane subfamily M, alpha member 1RELT-like 1 RELL1 226430_at unknown other 1.214 ring finger protein 182RNF182 230720_at unknown other 2.696 seizure related 6 homolog-likeSEZ6L 207873_x_at Plasma other 0.866 Membrane seizure related 6 homologSEZ6L 211894_x_at Plasma other 0.797 (mouse)-like Membrane similar tohCG2031213 LOC728052 1558795_at unknown other 2.239 single-mindedhomolog 1 SIM1 1556300_s_at Nucleus transcription 6.36 (Drosophila)regulator single-minded homolog 1 SIM1 206876_at Nucleus transcription5.737 (Drosophila) regulator SLIT and NTRK-like family, SLITRK5214930_at unknown other 4.094 member 5 SMG1 homolog, LOC641298 244766_atunknown other 1.065 phosphatidylinositol 3-kinase- related kinasepseudogene solute carrier family 44, SLC44A5 1569112_at unknown other2.738 member 5 solute carrier family 44, SLC44A5 235763_at unknown other1.845 member 5 solute carrier family 7 SLC7A2 225516_at Plasmatransporter 1.892 (cationic amino acid Membrane transporter, y+ system),member 2 solute carrier organic anion SLCO3A1 219229_at Plasmatransporter 1.622 transporter family, member Membrane 3A1spermatogenesis associated, SPATS2L 222154_s_at unknown other 0.907serine-rich 2-like synaptotagmin XIII SYT13 226086_at unknowntransporter −4.754 tissue factor pathway inhibitor 2 TFPI2 209278_s_atExtracellular other 2.326 Space transcription factor 7-like 2 (T- TCF7L2212761_at Nucleus transcription 1.005 cell specific, HMG-box) regulatortransformer 2 alpha homolog TRA2A 213593_s_at Nucleus other 1.12transmembrane inner ear TMIE 1553601_a_at unknown other −3.334transmembrane protein 178 TMEM178 229302_at unknown other 1.697 U2 smallnuclear RNA U2AF1 232141_at Nucleus other 0.776 auxiliary factor 1v-raf-1 murine leukemia viral RAF1 1557675_at Cytoplasm kinase 0.696oncogene homolog 1 zinc finger protein 217 ZNF217 203739_at Nucleustranscription 1.096 regulator zinc finger protein 662 ZNF662 228538_atunknown other −3.352 The log ratio represent log₂ values where 0.585 islog₂(1.5) which is a 1.5-fold difference, 1 is log₂(2) which is a 2-folddifference, 1.584 is log₂(3) which is a 3-fold difference, 2 is log₂(4)which is a 4-fold difference, 2.321 is log₂(5) which is a 5-folddifference, 2.584 is log₂(6) which is a 6-fold difference, 2.807 islog₂(7) which is a 7-fold difference, 3 is log₂(8) which is a 8-folddifference, 3.169 is log₂(9) which is a 9-fold difference, and 3.321 islog₂(10) which is a 10-fold difference.

Example VI Immuno-Based Method to Detect Picomolar Amounts of BoNT/A

The following example illustrates how to perform immuno-based methods ofdetecting BoNT/A activity that can detect picomolar amounts of theBoNT/A pharmaceutical product, such as, e.g., BOTOX® DYSPORT®/RELOXIN®,PURTOX®, XEOMIN®, NEURONOX®, or BTX-A.

1. Immuno-Based Method of Detecting BoNT/a Using ECL Sandwich ELISA.

To prepare a lysate from cells treated with a BoNT/A, approximately150,000 cells from an established clonal cell line was plated into thewells of 96-well tissue culture poly-D-lysine plates containing 100 μLof a serum-free medium containing Minimum Essential Medium, 2 mMGlutaMAX™ I with Earle's salts, 1× B27 supplement, 1×N2 supplement, 0.1mM Non-Essential Amino Acids, 10 mM HEPES and 60 μg/mL GT1b. The celllines used were BB10, H1, and SiMa. These cells were incubated in a 37°C. incubator under 5% carbon dioxide until the cells differentiated, asassessed by standard and routine morphological criteria, such as growtharrest and neurite extension (approximately 3 days). The media from thedifferentiated cells was aspirated from each well and replaced withfresh media containing one of the following: 1) SiMa cell line, 0(untreated), 0.98 U/mL, 1.9 U/mL, 3.91 U/mL, 7.81 U/mL, 15.6 U/mL, 31.3U/mL, 62.5 U/mL, 125 U/mL, 250 U/mL, or 500 U/mL of a BoNT/Apharmaceutical product reconstituted in a sodium chloride free medium;2) BB10, 0 (untreated), 0.49 U/mL, 0.98 U/mL, 1.9 U/mL, 3.91 U/mL, 7.81U/mL, 15.6 U/mL, 31.3 U/mL, 62.5 U/mL, 125 U/mL, 250 U/mL; 3) H1, 0(untreated), 0.3 U/mL, 1 U/mL, 3 U/mL, 9 U/mL, 28 U/mL, 83 U/mL, and 250U/mL. Because the BoNT/A pharmaceutical product contains sodiumchloride, its addition to the culture medium resulted in a hypertonicmedia that was detrimental to cell viability. To circumvent thehypertonicity issue, 200 μL of MEM media made without sodium chloridewas used to reconstitute the BoNT/A pharmaceutical product giving afinal concentration of 25 pM BoNT/A (500 U/mL). The matrix was keptconstant for all concentrations along the dose-response curve by addingsodium chloride in the media used to make the dilutions match the amountof excipients present at the highest concentration used (25 pM or 500U/mL). After a 24 hr treatment, the cells were washed, and incubated foran additional two days without toxin. To harvest the cells, 1×PBS wasaspirated, the cells lysed by adding 75 μL of Lysis Buffer comprising 50mM HEPES, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 1% Triton X-100 to eachwell, and the plate incubated on a shaker rotating at 500 rpm for 30minutes at 4° C. The plate was centrifuged at 4000 rpm for 20 minutes at4° C. to pellet cellular debris and the supernatant was transferred to acapture antibody coated 96-well plate to perform the detection step.

To prepare the α-SNAP-25 capture antibody solution, the α-SNAP-25monoclonal antibody contained in the ascites from hybridoma cell line2E2A6 (Example VII) was purified using a standard Protein A purificationprotocol. To prepare the α-SNAP-25 detection antibody solution,α-SNAP-25 rabbit polyclonal antibody S9684 (Sigma, St. Louis, Mo.) wasconjugated to Ruthenium(II)-tris-bipyridine-(4-methysulfonate) NHS esterlabeling reagent (Meso Scale Discovery, Gaithersburg, Md.) according tothe manufacturer's instructions (Meso Scale Discovery, Gaithersburg,Md.). The conjugation reaction was performed by adding 30 μL ofdistilled water reconstituted MSD SULFO-TAG™ stock solution to 200 μL of2 mg/mL α-SNAP-25 polyclonal antibodies and incubating the reaction atroom temperature for 2 hours in the dark. The labeled antibodies werepurified using a standard spin column protocol and the proteinconcentration determined using a standard colorimetric protein assay.The absorbance of the α-SNAP-25 antibody/MSD SULFO-TAG™ conjugate wasmeasured at 455 nm using a spectrophotometer to determine theconcentration in moles per liter. The detection antibody solution wasstored at 4° C. until needed.

To prepare the solid phase support comprising the capture antibody thatis specific for a SNAP-25 cleaved product, approximately 1 μL ofα-SNAP-25 monoclonal antibody 2E2A6 solution (45 μg/mL in 1×PBS, 750μg/mL of BSA) was added to each well of a 96-well MSD High Bind plate(Meso Scale Discovery, Gaithersburg, Md.) and the solution is allowed toair dry in a biological safety cabinet for 2-3 hours in order to liquidevaporate the solution, and then the plates were sealed and stored at 4°C. until needed. The dried capture antibody-bound wells were thenblocked by adding 150 μL of Blocking Buffer comprising 1×PBS, 0.05%Tween-20, 2% ECL Blocking reagent (GE Healthcare-Amersham), and 1% goatserum (Rockland Immunochemicals, Gilbertsville, Pa.) at room temperaturefor 2 hours with rotation at 600 rpm.

To detect the presence of a SNAP-25 cleavage product by ECL sandwichELISA analysis, the Blocking Buffer from plates was aspirated, 25 μL ofa lysate from cells treated with BoNT/A was added to each well and theplates were incubated at 4° C. for overnight. Plate wells were washedthree times by aspirating the cell lysate and rinsing each well threetimes with 200 μL 1×PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitanmonolaureate). After washing, 25 μl of 5 μg/mL α-SNAP-25 detectionantibody solution comprising 2% Amersham Blocking Reagent in 1×PBS, 0.1%TWEEN-20® (polyoxyethylene (20) sorbitan monolaureate) was added to eachwell, the plate was sealed, and the sealed plate was incubated at roomtemperature for 1 hour with shaking. After α-SNAP-25 detection antibodyincubation, the wells were washed three times with 250 μL 1×PBS, 0.1%TWEEN-20® (polyoxyethylene (20) sorbitan monolaureate). After washing150 μL of 1× Read Buffer (Meso Scale Discovery, Gaithersburg, Md.) wasadded to each well and the plates were read using a SECTOR™ Imager 6000Image Reader (Meso Scale Discovery, Gaithersburg, Md.). The raw data wasthen transferred to SigmaPlot v 10.0 and a 4-parameter logistics fit wasused to define the dose-response curves. There were no constraints usedfor the 4-parameter logistic function when plotting the data. Graphicalreports were generated using the following analysis: R2 (correlationcoefficient), a (Max for data set), b (hillslope), and X0±SE (EC₅₀value±standard error).

These results indicated that on average the cells comprising the BB10and H1 clonal cell lines were more susceptible to BoNT/A intoxication ascompared to cells comprising the parental SiMa cell line. Cells from aBB10 cell line exhibited an EC₅₀ for BoNT/A activity of 7 U/mL, cellsfrom a H1 cell line exhibited an EC₅₀ for BoNT/A activity of 8 U/mL, andcells from the parental SiMa cell line exhibited an EC₅₀ for BoNT/Aactivity of 13 U/mL. This method can also be performed in a multiplexfashion as described in Ester Fernandez-Salas, et al., Immuno-BasedBotulinum Toxin Serotype A Activity Assays, U.S. patent application Ser.No. 12/403,531, which is hereby incorporated by reference in itsentirety.

Example VII Transfection of BoNT/A Receptor DNA into Sensitive ClonalCell Lines Further Improves Sensitivity of the Clonal Cell Line forBoNT/A

The following example illustrates how to introduce a polynucleotidemolecule encoding a BoNT/A receptor into cells from a clonal cell lineto further improve susceptibility to BoNT/A intoxication or improveBoNT/A uptake capacity.

To introduce an exogenous BoNT/A receptor into cells comprising anestablished clonal cell line, a mammalian expression constructcomprising a polynucleotide molecule of SEQ ID NO: 139 encoding theFGFR3 of SEQ ID NO: 25, was transfected into cells from a H1 clonal cellline by a cationic lipid method. A suitable density (about 5×10⁶ cells)of cells from an established clonal cell line are plated in a 100 mmtissue culture dish containing 5 mL of complete culture media and grownin a 37° C. incubator under 5% carbon dioxide until the cells reached adensity appropriate for transfection. A 3 mL transfection solution isprepared by adding 1.5 mL of OPTI-MEM® Reduced Serum Medium containing60 μL of LIPOFECTAMINE® 2000 (Invitrogen, Carlsbad, Calif.) incubated atroom temperature for 5 minutes to 1.5 mL of OPTI-MEM® Reduced SerumMedium containing 24 μg of an expression construct encoding the FGFR3 ofSEQ ID NO: 25. This transfection mixture is incubated at roomtemperature for approximately 30 minutes. The complete media on thecells is replaced with the 3 mL transfection solution and the cells areincubated in a 37° C. incubator under 5% carbon dioxide forapproximately 8 hours. Transfection media is replaced with 3 mL of freshcomplete culture media and the cells are incubated in a 37° C. incubatorunder 5% carbon dioxide for approximately 24 hours. Media is replacedwith 3 mL of fresh complete culture media containing approximately 1 mMG418 (Invitrogen, Carlsbad, Calif.) for selection of transfected cells.Cells are incubated in a 37° C. incubator under 5% carbon dioxide forapproximately 1 week, the old media is replaced with fresh completeculture media containing 0.5 mM G418 and the cells are plated onto one96-well collagen IV coated plate at a plating density of 0.5 cells perwell. Cell growth and viability was monitored by microscopic examinationand after about 3 weeks single clones appeared in about 20% of thewells. These clones were grown for an additional 2 weeks and thentransferred to duplicate 24-well collagen IV coated plates, one tomaintain the clonal cell line and the other to test for BoNT/Asusceptibility.

To determine the susceptibility of the cells comprising the clonal celllines overexpressing a BoNT/A receptor, a Western blot analysis was doneto determine sensitivity and an ECL sandwich ELISA was done to determinean EC₅₀ value. A total of eight clones were tested for the FGFR3 stablytransfected H1 cells.

To prepare a cell lysate for the Western blot analysis, cells from theclonal cell line were plated into two wells in a poly-D-lysine coated96-well plate in serum-free medium containing Minimum Essential Medium,2 mM GLUTAMAX™ I with Earle's salts, 1× B27 supplement, 1×N2 supplement,0.1 mM Non-Essential Amino Acids, 10 mM HEPES and 25 μg/mL GT1b (AlexisBiochemicals, Lausen, Switzerland). These cells were incubated in a 37°C. incubator under 5% carbon dioxide until the cells differentiated, asassessed by standard and routine morphological criteria, such as growtharrest and neurite extension (approximately 3 days). The media from thedifferentiated cells was aspirated from each well and replaced withfresh media containing either 0 (untreated), 0.5 nM or 0.25 nM of aBoNT/A complex. After a 6 hr treatment, the cells were washed, andincubated for an additional 18 hr without toxin. After the incubation,the cells were washed by aspirating the growth media and rinsing eachwell with 200 μl of 1×PBS. To harvest the cells, the 1×PBS wasaspirated, the cells were lysed by adding 40 μL of 2×SDS Loading Bufferand the plate was swirled for an even coating of Loading Buffer. Thelysate was transferred to a new 96-well PCR plate (Axygen, Union City,Calif.) and the plate was heated to 95° C. for 5 min to complete celllysis and denature proteins.

To detect for the presence of both uncleaved SNAP-25 substrate andcleaved SNAP-25 products, an aliquot from each harvested sample wasanalyzed by Western blot. In this analysis, a 12 μL aliquot of theharvested sample was separated by MOPS polyacrylamide gelelectrophoresis using NUPAGE® Novex 12% Bis-Tris precast polyacrylamidegels (Invitrogen Inc., Carlsbad, Calif.) under denaturing, reducingconditions. Separated proteins were transferred from the gel ontonitrocellulose membranes (Bio-Rad Laboratories, Hercules, Calif.) byWestern blotting using a “Transfer +4 cassettes & Cooler” transferchamber (GE Healthcare, Piscataway, N.J.). Membranes were blocked byincubating at room temperature for 1 hr with gentle agitation in asolution containing Tris-Buffered Saline (TBS) (25 mM2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid (Tris-HCl) (pH7.4), 137 mM sodium chloride, 2.7 mM potassium chloride), 0.1% TWEEN-20®(polyoxyethylene (20) sorbitan monolaureate), 2% Bovine Serum Albumin(BSA), 5% nonfat dry milk. Blocked membranes were incubated at 4° C. forovernight in TBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitanmonolaureate), 2% BSA, and 5% nonfat dry milk containing a 1:5,000dilution of α-SNAP-25 rabbit polyclonal antiserum S9684 (Sigma, St.Louis, Mo.) as the primary antibody. The α-SNAP-25 rabbit polyclonalantibodies can detect both the uncleaved SNAP-25 substrate and theSNAP-25 cleavage product, allowing for the assessment of overall SNAP-25expression in each cell line and the percent of SNAP-25 cleaved afterBoNT/A treatment as a parameter to assess the amount of BoNT/A uptake.Primary antibody probed blots were washed three times for 5 minutes eachtime in TBS, TWEEN-20® (polyoxyethylene (20) sorbitan monolaureate).Washed membranes were incubated at room temperature for 2 hours in TBS,0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaureate), 2% BSA, and5% nonfat dry milk containing a 1:10,000 dilution of goat polyclonalanti-rabbit immunoglobulin G, heavy and light chains (IgG, H+L) antibodyconjugated to horseradish peroxidase (Invitrogen, Inc., Carlsbad,Calif.) as a secondary antibody. Secondary antibody-probed blots werewashed five times for 5 minutes each time in TBS, 0.1% TWEEN-20®(polyoxyethylene (20) sorbitan monolaureate). Signal detection of thelabeled SNAP-25 products were visualized using the ECL Plus™ WesternBlot Detection System (GE Healthcare, Amersham Biosciences, Piscataway,N.J.) and the membrane was imaged and the percent of cleaved SNAP-25products quantified with a Typhoon 9410 Variable Mode Imager and ImagerAnalysis software (GE Healthcare, Amersham Biosciences, Piscataway,N.J.). The choice of pixel size (100 to 200 pixels) and PMT voltagesettings (350 to 600, normally 400) depended on the individual blot. Thevolumes of the bands corresponding to either intact and cleaved SNAP-25product were quantified using Image Quant TL and the total amount ofSNAP-25 and the percentage of SNAP-25 cleaved were calculated based onthe intensity of these two SNAP-25 bands.

Clone H1 1.4 had a high percentage of SNAP-25 cleavage and high levelsof total SNAP25 and was selected for further testing. The selected clonewas first grown on a 10 cm² dish and then in T175 flasks to ensure thatenough cells were produced for both ECL sandwich ELISA assay and forfreezing stocks. Frozen stocks were made with 1×10⁶ cells and 1 mL ofRecovery Cell Culture Freezing Media (GIBCO-Invitrogen, #12648-010Freezing media).

To prepare a cell lysate for the ECL sandwich ELISA, approximately50,000 cells from H1 clonal cell line and from H1 1.4 clonal cell lineoverexpressing FGFR3 were plated and differentiated as described above.The media from the differentiated cells was aspirated from each well andreplaced with fresh media containing either (untreated), 0.27 pM, 0.82pM, 2.47 pM, 7.4 pM, 22.2 pM, 66.6 pM, or 200 pM of a BoNT/A complex ofa BoNT/A complex. After a 6 hr treatment, the cells were washed, andincubated overnight without toxin. After a 24 hr treatment, the cellswere washed by aspirating the BoNT/A containing media and rinsing eachwell with 200 μL of 1×PBS. Washed cells were harvested by lysing infreshly prepared Triton X-100 Lysis Buffer (150 mM NaCl, 20 mM Tris-HCl(pH 7.5), 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 plus proteaseinhibitors) at 4° C. for 30 minutes with constant agitation. Lysed cellswere centrifuged at 4000 rpm for 20 min at 4° C. to pellet debris.

The α-SNAP-25 capture antibody solution, the α-SNAP-25 detectionantibody solution, and the solid phase support comprising the captureantibody that is specific for a SNAP-25 cleaved product were prepared asdescribed in Example V. To detect the presence of a cleaved SNAP-25product by ECL sandwich ELISA, the plates were processed, and the datacollected and analyzed, and the EC₅₀ calculated as described in ExampleVI. The EC₅₀ for H1 1.4 cells was estimated to about 1.7 pM, compared toan EC₅₀ about 8.7 pM for H1 cells indicating that the newly generated H11.4 cell line is about 5 times more sensitive than the H1 cell line forBoNT/A uptake (average of two independent experiments with similarresults). The maximum signal for both cell lines was identicalindicating similar efficacy towards SNAP25 cleavage. The increase insensitivity is especially prominent at lower concentrations of BoNT/A,where the H1 1.4 cells are many times more sensitive to BoNT/A comparedto H1 cells producing a very robust signal over background at the 0.27pM concentration. Over-expression of FGFR3 was verified in a Westernblot using α-FGFR3 antibodies sc-123 (Santa Cruz Biotechnologies, SantaCruz, Calif.).

Example VIII Development of α-SNAP-25 Monoclonal Antibodies thatSelectively Bind a SNAP-25 Epitope Having a Free Carboxyl-terminus atthe P₁ Residue of the BoNT/A Cleavage Site Scissile Bond

The following example illustrates how to make α-SNAP-25 monoclonalantibodies that can selectively bind to a SNAP-25 epitope having acarboxyl-terminus at the P₁ residue of the BoNT/A cleavage site scissilebond.

1. Generation of α-SNAP-25 Monoclonal Antibodies.

α-SNAP-25 monoclonal antibodies with higher binding affinity for aSNAP-25 cleavage product and/or α-SNAP-25 antibodies with a higherbinding specificity for a SNAP-25 cleavage product were developed. Inaddition, because a permanent, stable and renewable source of α-SNAP-25antibodies is preferable for an FDA-approved assay, screens to isolatemonoclonal antibodies were undertaken. To develop monoclonal α-SNAP-25antibodies the 13-residue peptide CDSNKTRIDEANQ_(COOH) (SEQ ID NO: 38)was designed as a SNAP-25 antigen having a carboxyl-terminus at the P₁residue of the BoNT/A cleavage site scissile bond. This peptidecomprises a flexible linker region and a N-terminal Cysteine residue forconjugation to KLH and amino acids 186-197 of human SNAP-25 (SEQ ID NO:5) with a carboxylated C-terminal glutamine (SEQ ID NO: 38). Thegeneration of monoclonal antibodies to well-chosen, unique peptidesequences provides control over epitope specificity, allowing theidentification of a particular subpopulation of protein among a pool ofclosely related isoforms. Blast searches revealed that this peptide hashigh homology only to SNAP-25 and almost no possible cross-reactivitywith other proteins in neuronal cells. The sequence was also carefullyscrutinized by utilizing computer algorithms to determine hydropathyindex, protein surface probability, regions of flexibility, andfavorable secondary structure, followed by proper orientation andpresentation of the chosen peptide sequence. The peptide was synthesizedand conjugated to Keyhole Limpet Hemocyanin (KLH) to increaseimmunogenicity. Six Balb/c mice were immunized with this peptide, andafter three immunizations in about eight weeks, the mice were bled fortesting. The blood was allowed to clot by incubating at 4° C. for 60minutes. The clotted blood was centrifuged at 10,000×g at 4° C. for 10minutes to pellet the cellular debris. The resulting serum sample wasdispensed into 50 μL aliquots and stored at −20° C. until needed.

A similar strategy based on other SNAP-25 antigens disclosed in thepresent specification is used to develop α-SNAP-25 monoclonal antibodiesthat can selectively bind to a SNAP-25 having a carboxyl-terminus at theP₁ residue of the BoNT/A cleavage site scissile bond. For example, theSNAP-25 antigen of SEQ ID NO: 45 can be conjugated to KLH instead of theSNAP-25 antigen of SEQ ID NO: 38. As another example, the amino acids186-197 of human SNAP-25 from the SNAP-25 antigen of SEQ ID NO: 38 canbe replaced with SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ IDNO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 147, or SEQ ID NO: 148.

2. Screening for the Presence of α-SNAP-25 Monoclonal Antibodies.

To determine the presence of an α-SNAP-25 monoclonal antibody that canselectively bind to a SNAP-25 antigen having a carboxyl-terminus at theP₁ residue of the BoNT/A cleavage site scissile bond, comparative ELISAand cell-based cleavage assay were performed using the extracted mouseserum. For comparative ELISA, two fusion proteins were constructed:BIRA-HIS TAG®-SNAP-25₁₃₄₋₁₉₇ of SEQ ID NO: 48 and the BIRA-HISTAG®-SNAP-25₁₃₄₋₂₀₆ of SEQ ID NO: 49. BIRA-HIS TAG®-SNAP-25₁₃₄₋₁₉₇comprised a naturally-biotinylated 16 amino acid BirA peptide of SEQ IDNO: 50 amino-terminally linked to a SNAP-25 peptide comprising aminoacids 134-197 of SEQ ID NO: 5. BIRA-HIS TAG®-SNAP-25₁₃₄₋₂₀₆ comprised anaturally-biotinylated 16 amino acid BirA peptide of SEQ ID NO: 50amino-terminally linked to a SNAP-25 peptide comprising amino acids134-206 of SEQ ID NO: 5. These two substrates were suspended in 1×PBS ata concentration of 10 μg/mL BIRA-HIS TAG®-SNAP-25₁₃₄₋₁₉₇ and theBIRA-HIS TAG®-SNAP-25₁₃₄₋₂₀₆. The BIRA-HIS TAG®-SNAP-25₁₃₄₋₁₉₇ and theBIRA-HIS TAG®-SNAP-25₁₃₄₋₂₀₆ were coated onto separate plates by addingapproximately 100 μl of the appropriate Substrate Solution andincubating the plates at room temperature for one hour. Washed plateswere incubated at 37° C. for one hour in 0.5% BSA in 1×TBS containing a1:10 to 1:100 dilution of an antibody-containing serum derived from oneof the six immunized mice (Mouse 1, Mouse 2, Mouse 3, Mouse 4, Mouse 5,and Mouse 6). Primary antibody probed plates were washed four times for5 minutes each time in 200 μl TBS, 0.1% TWEEN-20® (polyoxyethylene (20)sorbitan monolaureate). Washed plates were incubated at 37° C. for 1hour in 1×TBS containing a 1:10,000 dilution of goat polyclonalanti-mouse IgG antibody conjugated to Horseradish peroxidase (PierceBiotechnology, Rockford, Ill.) as a secondary antibody. Secondaryantibody-probed plates were washed four times in 200 μl TBS, 0.1%TWEEN-20® (polyoxyethylene (20) sorbitan monolaureate). Chromogenicdetection of the labeled SNAP-25 products were visualized by chromogenicdetection using ImmunoPure TMB substrate kit (Pierce Biotechnology,Rockford, Ill.). The development of a yellow color in the BIRA-HISTAG®-SNAP-25₁₃₄₋₁₉₇ coated plates, but not the BIRA-HISTAG®-SNAP-25₁₃₄₋₂₀₆ coated plates, indicated that the α-SNAP-25 antibodypreferentially recognized the SNAP-25₁₉₇ cleavage product. The resultedindicated that of the six mice used for immunization, three mice (Mouse2, Mouse 3, and Mouse 4) had higher titers and more specificity towardsa SNAP-25 antigen having a carboxyl-terminus at the P₁ residue of theBoNT/A cleavage site scissile bond.

These results were confirmed using an ELISA light chain activity assay.A 96-well Reacti-Bind Streptavidin coated plates (Pierce Biotechnology,Rockford, Ill.) were prepared by adding approximately 100 μl of thefollowing Substrate Solution: Rows A-C were coated with 100 μL ofBIRA-HIS TAG®-SNAP-25₁₃₄₋₁₉₇ at twelve different concentrations; RowsD-H were coated with 100 μL of BIRA-HIS TAG®-SNAP-25₁₃₄₋₂₀₆ at 10 μg/mL.The plates were washed by aspirating the Substrate Solution and rinsingeach well three times with 200 μl TBS, 0.1% TWEEN-20® (polyoxyethylene(20) sorbitan monolaureate). Dilutions of BoNT/A were pre-reduced at 37°C. for 20 minutes in BoNT/A Incubation Buffer (50 mM HEPES, pH 7.4, 1%fetal bovine serum, 10 pM ZnCl₂, 10 mM dithiothrietol) and 100 μl of thepre-reduced BoNT/A was added to the substrate-coated plates andincubated at 37° C. for 90 minutes. BoNT/A treated plates were washed byaspirating the BoNT/A Incubation Buffer and rinsing each plate threetimes with 200 μl TBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitanmonolaureate). Washed plates were incubated at 37° C. for one hour in0.5% BSA in 1×TBS containing a 1:10 to 1:100 dilution of theantibody-containing serum being tested. Primary antibody probed plateswere washed four times for 5 minutes each time in 200 μl TBS, 0.1%TWEEN-20® (polyoxyethylene (20) sorbitan monolaureate). Washed plateswere incubated at 37° C. for 1 hour in 1×TBS containing a 1:10,000dilution of goat polyclonal anti-mouse IgG antibody conjugated toHorseradish peroxidase (Pierce Biotechnology, Rockford, Ill.) as asecondary antibody. Secondary antibody-probed plates were washed fourtimes in 200 μl TBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitanmonolaureate). Chromogenic detection of the labeled SNAP-25 productswere visualized by chromogenic detection using ImmunoPure TMB substratekit (Pierce Biotechnology, Rockford, Ill.). The development of a yellowcolor, which correlated with the presence of the SNAP-25₁₉₇ cleavageproduct was detected in BoNT/A treated samples, but not untreatedcontrols, using antibody-containing serum derived from all six immunizedmice (Mouse 1, Mouse 2, Mouse 3, Mouse 4, Mouse 5, and Mouse 6). Thus,the comparative ELISA analysis indicated that of the mice used forimmunization, three mice (Mouse 2, Mouse 3, and Mouse 4) had highertiters and more specificity towards a SNAP-25 antigen having acarboxyl-terminus at the P₁ residue of the BoNT/A cleavage site scissilebond.

For cell-based cleavage assay, a suitable density of PC12 cells wereplated into 60 mm² tissue culture plates containing 3 mL of anappropriate serum medium (Table 1). The cells were grown in a 37° C.incubator under 5% carbon dioxide until cells reached the appropriatedensity. A 500 μL transfection solution was prepared by adding 250 μL ofOPTI-MEM® Reduced Serum Medium containing 15 μL of LIPOFECTAINE® 2000(Invitrogen Inc., Carlsbad, Calif.) incubated at room temperature for 5minutes to 250 μL of OPTI-MEM® Reduced Serum Medium containing 10 μg ofa pQBI-25/GFP-BoNT/A-LC expression construct (SEQ ID NO: 51). ThepQBI-25/GFP-BoNT/A-LC expression construct comprises a pQBI-25expression vector (Qbiogene Inc., Carlsbad, Calif.) whose promoterelements are functionally linked to a polynucleotide encoding theGFP-BoNT/A light chain of SEQ ID NO: 52. This transfection mixture wasincubated at room temperature for approximately 20 minutes. The mediawas replaced with fresh unsupplemented media and the 500 μL transfectionsolution was added to the cells. The cells were then incubated in a 37°C. incubator under 5% carbon dioxide for approximately 6 to 18 hours.The cells were washed and harvested as described in Example II. Todetect for the presence of the cleaved SNAP-25₁₉₇ product, an aliquotfrom each harvested sample was analyzed by Western blot as described inExample II, except that the primary antibody used was a 1:1,000 dilutionof the antibody-containing serum and the secondary antibody used was a1:20,000 of mouse α-IgG Horseradish Peroxidase (Pierce Biotechnology,Rockford, Ill.). A single band corresponding to the SNAP-25₁₉₇ cleavageproduct was detected in BoNT/A treated samples, but not untreatedcontrols, using antibody-containing serum derived from three mice (Mouse2, Mouse 3, and Mouse 4). Thus, the cell-based cleavage assay indicatedthat of the mice used for immunization, three mice (Mouse 2, Mouse 3,and Mouse 4) had higher titers and more specificity towards a SNAP-25antigen having a carboxyl-terminus at the P₁ residue of the BoNT/Acleavage site scissile bond.

3. Production of Hybridomas.

To make hybridomas producing α-SNAP-25 monoclonal antibodies that canselectively bind to a SNAP-25 antigen having a carboxyl-terminus at theP₁ residue of the BoNT/A cleavage site scissile bond, the spleen fromMouse 2 was harvested three days subsequent to a final “booster”immunization and the spleen cells were fused with myeloma cells P3-X63Ag8.653 using standard hybridoma protocols. These cells were plated intofive 96-well plates and hybrids were selected using HAT medium. Within8-14 days after fusion, the first screening of the approximately 480parent clones was carried out using comparative ELISA with the BIRA-HISTAG®-SNAP-25₁₃₄₋₁₉₇ and the BIRA-HIS TAG®-SNAP-25₁₃₄₋₂₀₆ peptides coatedin two separate plates. The comparative ELISA provided a quick screenmethod to identify hybridomas producing antibodies specific for thecleaved SNAP-25₁₉₇. The top 18 clones were subjected to furtherscreening using the cell-based cleavage assay described above andimmunostaining of LC/A transfected cells. (Table 18).

TABLE 18 Analysis of Supernatants Containing α-SNAP-25 MonoclonalAntibody Comparative ELISA Cell-Based Assay Clone OD SNAP-25₁₉₇ ODSNAP-25₂₀₆ Ratio_(197/206) Ratio_(206/197) SNAP-25₁₉₇ SNAP-25₂₀₆ 1D31.805 0.225 8.02 0.13 +++ − 1F12 0.365 0.093 3.92 0.25 − − 1G10 0.5900.137 4.31 0.23 ++ − 1H1 0.335 0.121 2.77 0.36 − − 1H8 0.310 0.302 1.030.97 + − 2C9 0.139 0.274 0.51 1.97 − − 2E2 0.892 0.036 24.78 0.04 ++ −2E4 0.228 0.069 3.30 0.30 + − 2F11 1.095 1.781 0.61 1.63 − − 3C1 1.2680.053 23.92 0.04 ++ − 3C3 0.809 0.052 15.56 0.06 ++ − 3E1 0.086 0.1550.55 1.80 0 − 3E8 2.048 0.053 38.64 0.03 +++ − 3G2 0.053 0.158 0.34 2.98− − 4D1 0.106 0.218 0.49 2.06 − − 4G6 0.061 0.159 0.38 2.61 − − 5A50.251 0.106 2.37 0.42 + − 5F11 0.243 0.061 3.98 0.25 − −

Clones 1D3, 1G10, 2E2, 3C1, 3C3, and 3E8 were further cloned by limitingdilution because the conditioned media produced by these clonescomprised α-SNAP-25 antibodies with a preferential binding specificityhaving a ratio_(197/206) of at least 4:1 for the SNAP-25₁₉₇ cleavageproduct relative to the SNAP-25₂₀₆ uncleaved substrate and detected theSNAP-25₁₉₇-cleavage product using the cell-based cleavage assay and theimmunostaining of PC12 cells transfected with GFP-LC/A. Similarly clones2C9, 2F11, 3G2, 4D1 and 4G6 were further cloned by limiting dilutionbecause the conditioned media produced by these clones comprisedα-SNAP-25 antibodies with a preferential binding specificity having aratio_(206/197) of at least 1.5:1 for the SNAP-25₂₀₆ uncleaved substraterelative to the SNAP-25₁₉₇ cleavage product and detected theSNAP-25₂₀₆-uncleaved substrate using the cell-based cleavage assay.These single-cell derived clones were screened again using comparativeELISA, cell-based cleavage, and immunostaining to confirm their affinityand specificity, and the antibodies were isotyped using standardprocedures. Ascites were produced from clones 1D3B8 (IgM.k), 1G10A12(IgG3.k), 2C9B10 (IgG3.k), 2E2A6 (IgG3.k), 2F11B6 (IgM.k), 3C1A5(IgG2a.k), and 3C3E2 (IgG2a.k). Clone 3E8 stopped producing antibodiesduring the cloning process and could not be further evaluated.

4. Evaluation of Binding Specificity of α-SNAP-25 Monoclonal Antibodies.

To evaluate binding specificity of an α-SNAP-25 monoclonal antibody thatcan selectively bind to a SNAP-25 antigen having a carboxyl-terminus atthe P₁ residue of the BoNT/A cleavage site scissile bond, ascites fromclones 1D3B8, 1G10A12, 2C9B10, 2E2A6, 2F11B6, 3C1A5, and 3C3E2 were usedto detect SNAP-25 cleavage product using the cell-based activity assay,immunocytochemistry and immunoprecipitation.

For the cell-based activity assay, binding specificity was determined byanalyzing the ability of α-SNAP-25 antibody-containing ascites to detectthe uncleaved SNAP-25₂₀₆ substrate and the cleaved SNAP-25₁₉₇ product byWestern blot analysis. A suitable density of PC12 cells were plated into60 mm² tissue culture plates containing 3 mL of an appropriate serummedium, grown in a 37° C. incubator under 5% carbon dioxide until anappropriate cell density was reached, and transfected with the either atransfection solution lacking the pQBI-25/GFP-BoNT/A-LC expressionconstruct (untransfected cells) or a transfection solution containingthe pQBI-25/GFP-BoNT/A-LC expression construct (transfected cells) asdescribed above. The cells were washed and harvested as described inExample I. To detect for the presence of both the uncleaved SNAP-25₂₀₆substrate and the cleaved SNAP-25₁₉₇ product, an aliquot from eachharvested sample was analyzed by Western blot as described in Example I,except that the primary antibody used was a 1:100 dilution of theα-SNAP-25 monoclonal antibody-containing ascites and the secondaryantibody used was a 1:20,000 of α-mouse IgG conjugated to HorseradishPeroxidase (Pierce Biotechnology, Rockford, Ill.). In addition, threecommercially available mouse α-SNAP-25 monoclonal antibodies weretested. SMI-81 (Sternberger Monoclonals Inc., Lutherville, Md.), anα-SNAP-25 antibody the manufacturer indicates detects both the uncleavedSNAP-25₂₀₆ substrate and the cleaved SNAP-25₁₉₇ product, was used at a15,000 dilution according to the manufacturer's recommendations. MC-6050(Research & Diagnostic Antibodies, Las Vegas, Nev.), an α-SNAP-25antibody the manufacturer indicates detects both the uncleavedSNAP-25₂₀₆ substrate and the cleaved SNAP-25₁₉₇ product, was used at a1:100 dilution according to the manufacturer's recommendations. MC-6053(Research & Diagnostic Antibodies, Las Vegas, Nev.), an α-SNAP-25antibody the manufacturer indicates detects only the cleaved SNAP-25₁₉₇product, was used at a 1:100 dilution according to the manufacturer'srecommendations.

Table 19 indicates the α-SNAP-25 antibody-containing ascites thatdetected only the SNAP-25₁₉₇ cleavage product. The cell-based cleavageassay indicated that ascites produced from clones 1D3B8, 2C9B10, 2E2A6,3C1A5, and 3C3E2 synthesize an α-SNAP-25 monoclonal antibody having highbinding specificity for the SNAP-25₁₉₇ cleavage product that allows forthe selective recognition of this cleavage product relative to theSNAP-25₂₀₆ uncleaved substrate. Commercial antibody SMI-81 detected theSNAP-25₂₀₆ uncleaved substrate, but only poorly recognized theSNAP-25₁₉₇ cleavage product (Table 19). Surprisingly, commercialantibody MC-6050 only detected the SNAP-25₂₀₆ uncleaved substrate, andfailed to recognize the SNAP-25₁₉₇ cleavage product (Table 19). Evenmore surprisingly, commercial antibody MC-6050 only detected theSNAP-25₂₀₆ uncleaved substrate, and failed to recognize the SNAP-25₁₉₇cleavage product, even though the manufacturer advertises that thisantibody selectively detects the SNAP-25₁₉₇ cleavage product (Table 19).Thus, this analysis indicates that while 1D3B8, 2C9B10, 2E2A6, 3C1A5,and 3C3E2 exhibit suitable selectivity for the SNAP-25₁₉₇ cleavageproduct, 1G10A12 and 2F11B6 do not. In addition, commercial antibodiesSMI-81, MC-6050 and MC-6053 all are unsuitable for the immuno-basedmethods disclosed in the present application because all failed toselectivity detect the SNAP-25₁₉₇ cleavage product.

For immunocytochemistry analysis, binding specificity was determined byanalyzing the ability of α-SNAP-25 antibody-containing ascites to detectthe uncleaved SNAP-25₂₀₆ substrate and the cleaved SNAP-25₁₉₇ product byimmunostaining. See e.g., Ester Fernandez-Salas et al., Plasma MembraneLocalization Signals in the Light Chain of Botulinum Neurotoxin, Proc.Natl. Acad. Sci., U.S.A. 101(9): 3208-3213 (2004). A suitable density ofPC12 cells were plated, grown, and transfected with either atransfection solution lacking the pQBI-25/GFP-BoNT/A-LC expressionconstruct (untransfected cells) or a transfection solution containingthe pQBI-25/GFP-BoNT/A-LC expression construct (transfected cells) asdescribed above. The cells were washed in 1×PBS and fixed in 5 mL of PAFat room temperature for 30 minutes. Fixed cells were washed in phosphatebuffered saline, incubated in 5 mL of 0.5% Triton® X-100 (polyethyleneglycol octylphenol ether) in 1×PBS, washed in 1×PBS, and permeabilizedin 5 mL of methanol at −20° C. for six minutes. Permeabilized cells wereblocked in 5 mL of 100 mM glycine at room temperature for 30 minutes,washed in 1×PBS, and blocked in 5 mL of 0.5% BSA in 1×PBS at roomtemperature for 30 minutes. Blocked cells were washed in 1×PBS andincubated at room temperature for two hours in 0.5% BSA in 1×PBScontaining a 1:10 dilution of an ascites from a clonal hybridoma cellline being tested. Primary antibody probed cells were washed three timesfor 5 minutes each time in 1×PBS. Washed cells were incubated at roomtemperature for 2 hours in 1×PBS containing a 1:200 dilution of goatpolyclonal anti-mouse immunoglobulin G, heavy and light chains (IgG,H+L) antibody conjugated to ALEXA® FLUOR 568 (Invitrogen Inc., Carlsbad,Calif.) as a secondary antibody. Secondary antibody-probed cells werewashed three times for 5 minutes each time in 1×PBS. Washed cells wereprepared for microscopic examination by mounting in VECTASHIELD®Mounting Media (Vector Laboratories, Burlingame, Calif.) andcoverslipped. Images of signal detection were obtained with a Leicaconfocal microscope using appropriate laser settings. Table 19 indicatesthat the α-SNAP-25 antibody-containing ascites that specificallydetected the SNAP-25₁₉₇-cleavage product. The immunocytochemistryanalysis indicated that ascites produced from clones 1D3B8, 2C9B10,2E2A6, 3C1A5, and 3C3E2 synthesize an α-SNAP-25 monoclonal antibodyhaving high binding specificity for the SNAP-25₁₉₇ cleavage product thatallows for the preferential recognition of this cleavage productrelative to the SNAP-25₂₀₆ uncleaved substrate.

For immunoprecipitation analysis, binding specificity was determined byanalyzing the ability of Protein A (HiTrap™ Protein A HP Columns, GEHealthcare, Amersham, Piscataway, N.J.), purified α-SNAP-25 monoclonalantibodies to precipitate the uncleaved SNAP-25₂₀₆ substrate and thecleaved SNAP-25₁₉₇ product. See e.g., Chapter 8 Storing and PurifyingAntibodies, pp. 309-311, Harlow & Lane, supra, 1998a. A suitable densityof PC12 cells were plated, grown, and transfected with either atransfection solution containing a pQBI-25/GFP expression construct(control cells; SEQ ID NO: 53) or a transfection solution containing thepQBI-25/GFP-BoNT/A-LC expression construct (experimental cells) asdescribed above. The pQBI-25/GFP expression construct comprises anexpression vector whose promoter elements are functionally linked to apolynucleotide encoding GFP of SEQ ID NO: 54. After an overnightincubation, the cells were washed by aspirating the growth media andrinsing each well with 200 μL 1×PBS. To harvest the cells, the PBS wasaspirated, the cells were lysed by adding an Immunoprecipitation LysisBuffer comprising 50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGDT, 10%glycerol, 1% Triton® X-100 (polyethylene glycol octylphenol ether) and a1× COMPLETE™ Protease inhibitor cocktail (Roche Applied Biosciences,Indianapolis, Ind.) and incubating at 4° C. for one hour. The lysedcells were centrifuged at 3,000×g at 4° C. for 10 minutes to removecellular debris and the supernatant transferred to a clean tube anddiluted to a protein concentration of approximately 1 mg/mL.Approximately 5 μg of purified monoclonal antibody was added to 0.5 mLof diluted supernatant and incubated at 4° C. for two hours. Afterprimary antibody incubation, approximately 50 μl of immobilized ProteinG (Pierce Biotechnology, Rockford, Ill.) was added to the dilutedsupernatant and incubated at 4° C. for one hour. The incubatedsupernatant was washed three times for 30 minutes each time by adding0.5 mL of Immunoprecipitation Lysis Buffer, centrifuging at 300×g at 4°C. for one minute to pellet the immobilized Protein G, and decanting thesupernatant. After washing, the pellet was resuspended in 30 μL of 1×SDSLoading Buffer and the sample was heated to 95° C. for 5 minutes. Todetect for the presence of both the uncleaved SNAP-25₂₀₆ substrate andthe cleaved SNAP-25₁₉₇ product, an aliquot from each harvested samplewas analyzed by Western blot as described in Example I, except that theprimary antibody used was a 1:1,000 dilution of the α-SNAP-25 polyclonalantibody serum (see Example IV) and the secondary antibody used was a1:20,000 of rabbit α-IgG Horseradish Peroxidase (Pierce Biotechnology,Rockford, Ill.). Table 19 indicates the α-SNAP-25 antibody-containingascites that specifically pulled down the SNAP-25₁₉₇-cleavage product byimmunoprecipitation analysis. The immunoprecipitation analysis indicatedthat ascites produced from clones 2E2A6 and 3C1A5 synthesize anα-SNAP-25 monoclonal antibody having high binding specificity for theSNAP-25₁₉₇ cleavage product that allows for the preferential recognitionof this cleavage product relative to the SNAP-25₂₀₆ uncleaved substrate.

TABLE 19 Analysis of Clone Ascites Containing α-SNAP-25 MonoclonalAntibody Cell-Based Assay Immunocytochemistry Immunoprecipitation CloneSNAP-25₁₉₇ SNAP-25₂₀₆ SNAP-25₁₉₇ SNAP-25₂₀₆ SNAP-25₁₉₇ SNAP-25₂₀₆ 1D3B8++ − ++ − Not Tested Not Tested 1G10A12 ++ ++ Not Tested Not Tested NotTested Not Tested 2C9B10 ++ − ++ − Not Tested Not Tested 2E2A6 ++ − ++ −++ − 2F11B6 + + + + Not Tested Not Tested 3C1A5 ++ − ++ − ++ − 3C3E2 + −Not Tested Not Tested Not Tested Not Tested MC-6050 − + Not Tested NotTested Not Tested Not Tested MC-6053 − + Not Tested Not Tested NotTested Not Tested SMI-81 −/+ ++ Not Tested Not Tested Not Tested NotTested

5. Evaluation of Binding Affinity of α-SNAP-25 Monoclonal Antibodies.

To determine the binding affinity of an α-SNAP-25 monoclonal antibodyshowing high binding specificity for either the SNAP-25₁₉₇ cleavageproduct or the SNAP-25₂₀₆ uncleaved substrate, binding affinity assayswere performed on a BIACORE 3000 instrument using carboxymethyl dextran(CM5) sensor chips (BIAcore, Inc., Piscataway, N.J.). Runs wereconducted at 25° C. with HBS-EP buffer comprising 10 mM HEPES (pH 7.4),150 mM sodium chloride, 3 mM EDTA, 0.005% (v/v) surfactant P20 at a flowrate of 10 μl/min. SNAP-25 peptides comprising amino acids 134-197 ofSEQ ID NO: 5 (SNAP-25₁₃₄₋₁₉₇) or amino acids 134-206 of SEQ ID NO: 5(SNAP-25₁₃₄₋₂₀₆) were covalently attached to the surface of the CM5sensor chips using standard amine coupling. Briefly, the CM5 chips wereactivated by a 7 minute injection of a mixture of 0.2 M1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.05 MN-hydroxysuccimide; the SNAP-25 peptides were then injected in 10 mMsodium acetate (pH 4.0) for 20 min at a flow rate of 10 μL/min; andunreacted succimide esters were blocked by a 7-min injection of 1 Methanolamine hydrochloride, pH 8.5. The immobilized amount ofSNAP-25₁₃₄₋₁₉₇ or SNAP-25₁₃₄₋₂₀₆ on the chip was reflected by a 100-150increase in response units (about 0.10-0.15 ng/mm²). Antibody samplescomprising either ascites or purified monoclonal antibodies producedfrom clones 1D3B8, 2C9B10, 2E2A6, 3C1A5, and 3C3E2, as well as,commercially available α-SNAP-25 antibodies were passed over the surfaceof the CM5 chips allowing an association time of 10 min and adissociation time of 20 min. The surfaces were regenerated between runsby a 1 minute injection of 10 mM glycine-HCl (pH 2.5) at a flow rate of15 μL/min. Sensorgram curves were fitted to a 1:1 kinetic binding modelwith the BIAevaluation 3.0 software.

The results indicate that both 2E2A6 and 3C1A5 were highly specific forcleaved SNAP-25₁₉₇ product over SNAP-25 uncleaved substrate (Table 20).When compared to the binding affinities of MC-6050 and MC-6053, 1D3B6had an approximately 10-fold higher equilibrium disassociation constantfor the SNAP-25 cleavage product relative to these commercial antibodies(Table 20). Interestingly, 2E2A6 had only a slightly lower equilibriumdisassociation constant for the SNAP-25 cleavage product relative tothese commercial antibodies (0.405 nM versus 0.497 and 0.508) (Table20). As neither of these commercial α-SNAP-25 antibodies selectivelyrecognized the SNAP-25 cleavage product (Table 19), an equilibriumdisassociation constant lower than about 0.5 nM appears, in part,critical to achieve such selectivity. Similarly, when compared to thebinding affinities of MC-6050 and MC-6053, 2E2A6 had an about at leastone-fold slower off rate/dissociation constant (6.74×10⁻⁵ versus8.82×10⁻⁴ s⁻¹ and 1.18×10⁻³ s⁻¹) (Table 20). This further suggests thatan off rate/dissociation constant lower than about 8.82×10⁻⁴ appears, inpart, critical to achieve selective binding for the SNAP-25 cleavageproduct. This result is consistent with 1 D3B8, which had an offrate/dissociation constant of 5.78×10⁻⁵ s⁻¹ (Table 20).

TABLE 20 Analysis of Binding Affinity α-SNAP-25 Monoclonal AntibodiesSPR 1D3B8 2E2A6 Parameter SNAP-25₁₉₇ SNAP-25₂₀₆ ^(a) SNAP-25₁₉₇SNAP-25₂₀₆ ^(b) Ka 1.06 × 10⁶ — 1.70 × 10⁶   — (M⁻¹ s⁻¹) (1.66 × 10⁵  )(—) Kd (s⁻¹) 5.78 × 10⁻⁵ — 1.53 × 10⁻⁴ — (6.74 × 10⁻⁵) (—) KD (nM) 0.050— 0.090 — (0.405) (—) SPR 3C1A5 2C9B10 Parameter SNAP-25₁₉₇ SNAP-25₂₀₆^(c) SNAP-25₁₉₇ SNAP-25₂₀₆ ^(d) Ka 2.17 × 10⁵ — 1.15 × 10⁴   — (M⁻¹ s⁻¹)Kd (s⁻¹) 2.88 × 10⁻⁴ — 3.11 × 10⁻⁴ — KD (nM) 1.33 — 27.1 — SPR MC-6050MC-6053 Parameter SNAP-25₁₉₇ SNAP-25₂₀₆ SNAP-25₁₉₇ SNAP-25₂₀₆ Ka 1.78 ×10⁶ 3.06 × 10² 2.32 × 10⁶   1.06 × 10²   (M⁻¹ s⁻¹) Kd (s⁻¹) 8.82 × 10⁻⁴6.07 × 10⁻³ 1.18 × 10⁻³ 2.56 × 10⁻⁵ KD (nM) 0.497 19,800 0.508 240^(a)No binding was observed when up to 125 nM of α-SNAP-25 monoclonalantibody 1D3B8 was passed over the surface of the CM5 sensor chip aftera 10 minute association time. ^(b)No binding was observed when up to 10μM of α-SNAP-25 monoclonal antibody 2E2A6 was passed over the surface ofthe CM5 sensor chip after a 10 minute association time. ^(c)No bindingwas observed when up to 100 nM of α-SNAP-25 monoclonal antibody 3C1A5was passed over the surface of the CM5 sensor chip after a 10 minuteassociation time. ^(d)No binding was observed when up to 100 nM ofα-SNAP-25 monoclonal antibody 2C9B10 was passed over the surface of theCM5 sensor chip after a 10 minute association time.

To compare the six different antibodies, the on-rate (ka) and off-rate(kd) for each was normalized using a program from the BIA evaluation 4.1software. For comparison of the on-rates, the data were firstindividually trimmed by deleting the re-generation portion and theinjection spikes, and then normalized to a 0 to 100 scale. Forcomparison of the off-rate, the data were normalized to the injectionstop/top point. This analysis showed that 2C9B10 had a much sloweron-rate than the other antibodies (FIG. 3A), and that MC-6053 has a muchfaster off-rate (dissociation) that the other antibodies (FIG. 3B). Thefast off-rate of MC-6053 indicates that this antibody will not performwell in the methods disclosed in the present specification because thisantibody will have difficulty staying bound to the substrate antigenduring the washing steps.

6. Sequencing of the Epitope from Isolated α-SNAP-25 MonoclonalAntibodies.

To determine the epitope of an isolated α-SNAP-25 monoclonal antibodythat can selectively bind to a SNAP-25 antigen having acarboxyl-terminus at the P₁ residue of the BoNT/A cleavage site scissilebond, the polynucleotide molecule encoding the variable heavy (V_(H))and variable light (V_(L)) chains of the α-SNAP-25 monoclonal antibodyproduced by hybridomas 1D3B8, 2C9B10, 2E2A6, 3C1A5 and 3C3E2 weresequenced. mRNA was extracted and purified from each hybridoma usingstandard protocols and reversed transcribed into cDNA using either anoligo dT anti-sense primer or a gene-specific (murine IgG1 CH and kappaCL) anti-sense primer. Specific murine and human constant domain primerswere used to amplify the cDNA by PCR after cDNA production to determinethe isotype of the antibody. Degenerate V_(H) and V_(L) primers wereused to amplify the variable domains from the cDNA. For 5′RACE, ahomopolymeric dCTP tail was added to the 3′ end of the cDNA. The heavyand light chains were then amplified with an oligo dG sense primer and agene specific (CH/KC) anti-sense primer. PCR products included thesequence of the signal peptide, variable domains and constant domains upto the anti-sense primer. The PCR products were gel purified to removesmall fragments, and cloned into a blunt or TA vector for sequencing.Five independent clones for each chain were sequenced and alignments ofV_(H) and V_(L) chains and consensus sequences were determined. Methodsused to determine the V_(H) and V_(L) amino acid sequences are describedin, e.g., Roger A. Sabbadini, et al., Novel Bioactive Lipid Derivativesand Methods of Making and Using Same, U.S. Patent Publication2007/0281320; and Peter Amersdorfer, et al., Molecular Characterizationof Murine Humoral Immune Response to Botulinum Neurotoxin Type A BindingDomain as Assessed by Using Phage Antibody Libraries, 65(9) Infect.Immun. 3743-3752, each of which is hereby incorporated by reference inits entirety. In addition, commercial services are available to sequencethe variable heavy (V_(H)) and variable light (V_(L)) chains of anantibody and identify the CDR regions, see, e.g., Fusion AntibodiesLtd., Northern Ireland.

The polynucleotide sequence comprising the V_(H) and V_(L) chains of theα-SNAP-25 monoclonal antibody produced by the hybridomas disclosed inthe present specification is as follows: 1 D3B8 V_(H) (SEQ ID NO: 71),2C9B10 V_(H) (SEQ ID NO: 73), 2E2A6 V_(H) (SEQ ID NO: 75), 3C1A5 V_(H)(SEQ ID NO: 77), 3C3E2 V_(H) variant 1 (SEQ ID NO: 79), 3C3E2 V_(H)variant 2 (SEQ ID NO: 81), 3C3E2 V_(H) variant 3 (SEQ ID NO: 149), 1D3B8V_(L) (SEQ ID NO: 83), 2C9B10 V_(L) (SEQ ID NO: 85), 2E2A6 V_(L) (SEQ IDNO: 87), 3C1A5 V_(L) (SEQ ID NO: 89), and 3C3E2 V_(L) (SEQ ID NO: 91).The amino acid sequence comprising the V_(H) and V_(L) chains of theα-SNAP-25 monoclonal antibody produced by the hybridomas disclosed inthe present specification is as follows: 1D3B8 V_(H) (SEQ ID NO: 72),2C9B10 V_(H) (SEQ ID NO: 74), 2E2A6 V_(H) (SEQ ID NO: 76), 3C1A5 V_(H)(SEQ ID NO: 78), 3C3E2 V_(H) variant 1 (SEQ ID NO: 80), 3C3E2 V_(H)variant 2 (SEQ ID NO: 82); 3C3E2 V_(H) variant 2 (SEQ ID NO: 150), 1D3B8V_(L) (SEQ ID NO: 84), 2C9B10 V_(L) (SEQ ID NO: 86), 2E2A6 V_(L) (SEQ IDNO: 88), 3C1A5 V_(L) (SEQ ID NO: 90), and 3C3E2 V_(L) (SEQ ID NO: 92).The amino acid sequences comprising the V_(H) and V_(L) CDR domains ofthe α-SNAP-25 monoclonal antibody produced by the hybridomas 1 D3B8,2C9B10, 2E2A6, 3C1A5, and 3C3E2 are given in Table 21.

TABLE 21 CDR Sequences of V_(H) and V_(L) domains from α-SNAP-25Monoclonal Antibodies SEQ ID CDR Sequence Identified In NO: V_(H) CDR 1TFTDHSIH 2E2A6 93 2C9B10 3C1A5 V_(H) CDR 1 TFTNYVIH 3C3E2 94 V_(H) CDR 1IFTDHALH 1D3B8 95 V_(H) CDR 2 YIFPGNGNIEYNDKFKG 2E2A6 96 V_(H) CDR 2YLFPGNGNFEYNEKFKG 2C9B10 97 3C1A5 V_(H) CDR 2 YINPYNDGSKYNEKFKG 3C3E2 98V_(H) CDR 2 YIFPGNGNIEYNEKFKG 1D3B8 99 V_(H) CDR 3 KRMGY 2E2A6 100 3C1A5V_(H) CDR 3 KKMDY 2C9B10 101 1D3B8 V_(H) CDR 3 ARMDY 3C3E2var1 102V_(H) CDR 3 ARMGY 3C3E2var2 151 V_(H) CDR 3 ARHLANTYYYFDY 3C3E2var3 152V_(L) CDR 1 RSSQSIVHSNGNTYLE 1D3B8 103 V_(L) CDR 1 RTTENIYSYFV 2C9B10104 V_(L) CDR 1 KSSQSLLYTNGKTYLT 2E2A6 105 V_(L) CDR 1 KSSQSLLNTNGKTYLT3C1A5 106 V_(L) CDR 1 RASQNIGNYLH 3C3E2 107 V_(L) CDR 2 KVSNRFS 1D3B8108 V_(L) CDR 2 NAKSLAE 2C9B10 109 V_(L) CDR 2 LVSELDS 2E2A6 110V_(L) CDR 2 LVSKLDS 3C1A5 111 V_(L) CDR 2 YASQSIS 3C3E2 112 V_(L) CDR 3FQGSHVPPT 1D3B8 113 V_(L) CDR 3 QHHYGTPYT 2C9B10 114 V_(L) CDR 3LQSAHFPFT 2E2A6 115 V_(L) CDR 3 LQSSHFPFT 3C1A5 116 V_(L) CDR 3QQSDTWPLT 3C3E2 117

Non-limiting examples of amino acid sequences comprising V_(H) CDRdomain variants of the α-SNAP-25 monoclonal antibody produced by thehybridomas disclosed in the present specification include V_(H) CDR1variant SEQ ID NO: 118 for 1D3B8; V_(H) CDR1 variant SEQ ID NO: 119 for2C9B10, 2E2A6 and 3C1A5 V_(H); V_(H) CDR1 variant SEQ ID NO: 120 for3C1A5 V_(H) and 3C3E2 variant 3; V_(H) CDR2 variant SEQ ID NO: 121 for1D3B8 and 2E2A6; V_(H) CDR2 variant SEQ ID NO: 122 for 2C9B10 and 3C1A5V_(H); V_(H) CDR2 variant SEQ ID NO: 123 for 3C1A5 V_(H) and 3C3E2variant 3; V_(H) CDR3 variant MDY for 1D3B8 and 2C9B10; V_(H) CDR3variant MGY for 2E2A6 and 3C1A5 V_(H); and V_(H) CDR3 variant SEQ ID NO:124 for 3C1A5 V_(H) and 3C3E2 variant 3. Non-limiting examples of aminoacid sequences comprising V_(L) CDR domain variants of the α-SNAP-25monoclonal antibody produced by the hybridomas disclosed in the presentspecification include V_(L) CDR1 variant SEQ ID NO: 125 for 1 D3B8;V_(L) CDR1 variant SEQ ID NO: 126 for 2C9B10; V_(L) CDR1 variant SEQ IDNO: 127 for 2E2A6; V_(L) CDR1 variant SEQ ID NO: 128 for 3C1A5; V_(L)CDR1 variant SEQ ID NO: 129 for 3C3E2; V_(L) CDR2 variant KVS for 1D3B8;V_(L) CDR2 variant NAK for 2C9B10; V_(L) CDR2 variant LVS for 2E2A6;V_(L) CDR2 variant YAT for 3C1A5; and V_(L) CDR2 variant YAS for 3C3E2.

Example IX Development of α-SNAP-25 Polyclonal Antibodies thatSelectively Bind a SNAP-25 Epitope Having a Free Carboxyl-terminus atthe P₁ Residue of the BoNT/A Cleavage Site Scissile Bond

The following example illustrates how to make α-SNAP-25 polyclonalantibodies that can selectively bind to a SNAP-25 epitope having acarboxyl-terminus at the P₁ residue of the BoNT/A cleavage site scissilebond.

To develop α-SNAP-25 polyclonal antibodies that bind an epitopecomprising a carboxyl-terminus at the P1 residue from the BoNT/Acleavage site scissile bond from a SNAP-25 cleavage product, the10-residue peptide CGGGRIDEANQ (SEQ ID NO: 46) was designed as a SNAP-25cleavage product antigen. This peptide comprising a N-terminal Cysteineresidue for conjugation to KLH, a G-spacer flexible spacer (GGG) linkedto amino acids 191-197 of human SNAP-25 (SEQ ID NO: 5) and has acarboxylated C-terminal glutamine. Blast searches revealed that thispeptide has high homology only to SNAP-25 and almost no possiblecross-reactivity with other proteins in neuronal cells. The sequence wasalso carefully scrutinized by utilizing computer algorithms to determinehydropathy index, protein surface probability, regions of flexibility,and favorable secondary structure, followed by proper orientation andpresentation of the chosen peptide sequence. The peptide was synthesizedand conjugated to Keyhole Limpet Hemocyanin (KLH) to increaseimmunogenicity. Before the animals were immunized, naïve rabbits werefirst screened against cell lysates from candidate cell lines in aWestern blot in order to identify animals that had no immunoreactivityto the proteins present in the cell lysates. Two pre-screened rabbitswere immunized with this peptide, and after three immunizations in abouteight weeks, the rabbits were bled for testing. The blood was allowed toclot by incubating at 4° C. for 60 minutes. The clotted blood wascentrifuged at 10,000×g at 4° C. for 10 minutes to pellet the cellulardebris. The resulting serum sample was dispensed into 50 μL aliquots andstored at −20° C. until needed.

A similar strategy based on other SNAP-25 antigens disclosed in thepresent specification is used to develop α-SNAP-25 polyclonal antibodiesthat bind an epitope comprising a carboxyl-terminus at the P1 residuefrom the BoNT/A cleavage site scissile bond from a SNAP-25 cleavageproduct. For example, the SNAP-25 antigen of SEQ ID NO: 47 can beconjugated to KLH instead of the SNAP-25 antigen of SEQ ID NO: 46. Asanother example, the amino acids 191-197 of human SNAP-25 from theSNAP-25 antigen of SEQ ID NO: 38 can be replaced with SEQ ID NO: 33, SEQID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39,SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ IDNO: 44.

2. Screening for the Presence of α-SNAP-25 Polyclonal Antibodies.

To determine the presence of α-SNAP-25 polyclonal antibodies that canselectively bind to a SNAP-25 antigen having a carboxyl-terminus at theP₁ residue of the BoNT/A cleavage site scissile bond, comparative ELISAand cell-based cleavage assays were performed using the extracted rabbitserum as described in Example VIII. The serum from both rabbitscontained α-SNAP-25 polyclonal antibodies that can selectively bind to aSNAP-25 antigen having a carboxyl-terminus at the P₁ residue of theBoNT/A cleavage site scissile bond. The α-SNAP-25 rabbit polyclonalantibodies were designated as NTP 22 and NTP 23.

3. Purification of α-SNAP-25 Polyclonal Antibodies.

To purify α-SNAP-25 polyclonal antibodies that can selectively bind to aSNAP-25 antigen having a carboxyl-terminus at the P₁ residue of theBoNT/A cleavage site scissile bond, NTP 22 and NTP 23 antibodies fromrabbit serum were purified using affinity columns containing the SNAP-25antigen of SEQ ID NO: 46.

4. Evaluation of Binding Specificity of α-SNAP-25 Polyclonal Antibodies.

To evaluate binding specificity of an α-SNAP-25 polyclonal antibody thatcan selectively bind to a SNAP-25 antigen having a carboxyl-terminus atthe P₁ residue of the BoNT/A cleavage site scissile bond, purified NTP22 and NTP 23 α-SNAP-25 polyclonal antibodies were used to detectcleavage product using the cell-based activity assay,immunnocytochemistry and immunoprecipitation as described in ExampleVIII. The cell-based cleavage assay, immunocytochemistry analysis andimmunoprecipitation analysis all indicated that NTP 22 and NTP 23α-SNAP-25 polyclonal antibodies did not cross-react with uncleavedSNAP-25. Thus both NTP 22 and NTP 23 have high binding specificity forthe SNAP-25₁₉₇ cleavage product that allows for the preferentialrecognition of this cleavage product relative to the SNAP-25₂₀₆uncleaved substrate. Affinity for the antigens can be determined usingSPR in the BiAcore as described in Example VIII.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the invention are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

1. An established single cell derived clonal cell line comprising cellssusceptible to intoxication by botulinum toxin type A (BoNT/A), whereinthe cells exhibit at least a 1.5-fold increase in mRNA expression levelsafter differentiation in the genes ALCAM, AURKA, E2F, ERK, ESPL1, HMMR,KIF15, KIF22, MKI67, 01P5, PHLPP, PP1/PP2A, PPP1R3C, PTTG1, RB and TPX2relative to the mRNA expression levels of each of these genes in cellsfrom the parental SiMa cell line DSMZ ACC 164; and, wherein the cellsexhibit at least a 1.5-fold decrease in mRNA expression levels afterdifferentiation in the genes KISSR and SCN2A as compared to the mRNAexpression levels of each of these genes in cells from the parental SiMacell line DSMZ ACC 164; and, wherein the clonal cell line comprisescells susceptible to BoNT/A intoxication by about 100 pM or less of aBoNT/A.
 2. The established clonal cell line of claim 1, wherein thecells exhibit at least a 2.0-fold difference in gene expression levelsof at least one gene as compared to the expression levels of the gene incells from the parental SiMa cell line DSMZ ACC
 164. 3. The establishedclonal cell line of claim 1, wherein the cells exhibit at least a3.0-fold difference in gene expression levels of at least one gene ascompared to the expression levels of the gene in cells from the parentalSiMa cell line DSMZ ACC
 164. 4. The established clonal cell line ofclaim 1, wherein the cells exhibit at least a 4.0-fold difference ingene expression levels of at least one gene as compared to theexpression levels of the gene in cells from the parental SiMa cell lineDSMZ ACC
 164. 5. The established clonal cell line of claim 1, whereinthe cell from an established clonal cell line exhibits about 100 pM orless of a BoNT/A for at least 5 or more cell passages.